JP2004141074A - Method for easily creating knockout animal - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a system and a method for elucidating the function of a great number of genes at an individual level of animals using minimal labor and time. <P>SOLUTION: A method for creating a knockout animal comprises the step(1) of carrying out a gene trap using embryonic stem cells and a gene trap vector, the step(2) of preparing an oligonucleotide corresponding to the trapped gene from each of the embryonic stem cells, the step(3) of making a microarray using the thus prepared oligonucleotide, the step(4) of offering genetic information on the oligonucleotide using the microarray, the step(5) of selecting the gene having desired properties based on the genetic information, and the step(6) of creating the objective knockout animal using the embryonic stem cells where the genes having desired properties are destructed. <P>COPYRIGHT: (C)2004,JPO

Description

の反応溶液を調製して、６５℃で、５分間インキュベートして、次に氷上に３分以上放置する。次に、

の反応液を調製して、３７℃で５０分間、次に７２℃で１５分間インキュベートする。１０ｍＭのＴｒｉｓ−ＨＣｌ（ｐＨ８．０）を８０μｌ添加し、そして−２０℃で保存する。
【０１８２】
（実施例７；３’ＲＡＣＥ）
各ウェルに対応する総ＲＮＡから調製したｃＤＮＡを用いて、その３’末端を３’ＲＡＣＥ法によって調製する（Ｏｈａｒａ，Ｏ．ｅｔ　ａｌ．（１９８９）ＰＮＡＳ　８６，５６７３−５６７７）。
【０１８３】
３’ＲＡＣＥ法に使用するポリメラーゼとして、Ａｄｖａｎｔａｇｅ−ＧＣ２ポリメラーゼミックス（ＢＤ　Ｂｉｏｓｃｉｅｎｃｅｓ　Ｃｌｏｎｅｔｅｃｈ、Ｐａｌｏ　Ａｌｔｏ，ＣＡ，ＵＳＡ）を使用して、以下の反応を行う。
【０１８４】
１）１回目のＲＡＣＥ反応

の反応溶液を調製して、以下のサイクル：

を行い、反応液を４℃で保存する。
【０１８５】
２）２回目のＲＡＣＥ反応

の反応溶液を調製して、以下のサイクル：

を行い、反応液を４℃で保存する。
【０１８６】
（実施例８；マイクロアレイの調製）
実施例７において３’ＲＡＣＥ法で調製したＤＮＡフラグメントの塩基配列を直接配列決定法（Ｋｕｓｕｋａｗａ，Ｎ．ｅｔ　ａｌ．１９９０，ＢｉｏＴｅｃｈｎｉｑｕｅｓ　９：６６−７２）によって決定した。直接配列決定には、ＮＥＯ３．５プライマーを用いた。この配列情報に基づいて各遺伝子に特異的なプライマー（上流向き、つまりアンチセンス）を合成する。そしてそれぞれの遺伝子断片を、ＮＥＯ３．５プライマーおよび新たに合成した遺伝子特異的なプライマーを用いて、ＰＣＲによって増幅しなおした。マイクロアレイへの固定は、この増幅しなおしたＤＮＡフラグメントを用いた。
【０１８７】
増幅しなおしたＤＮＡフラグメントの吸光度（ＯＤ２６０）を測定し、それぞれのフラグメントの長さを考慮した上で、各フラグメントの濃度を０．５−１．５ｐｍｏｌ／μｌに合わせた。その上で各ＤＮＡフラグメントを９６ウェルプレートに移した。
【０１８８】
マイクロアレイの作製には、マルチピンプロッター９６（ＡＢ−６６９０ＮＭ型、アトー株式会社、東京）およびバイオダインＢナイロン膜（Ｐａｌｌ　Ｇｅｌｍａｎ　Ｌａｂｏｒａｔｏｒｙ，Ａｎｎ　Ａｒｂｏｒ，ＭＩ，ＵＳＡ）を用いた。１１．０×７．５ｃｍのメンブレンに８６４種類のＤＮＡフラグメントをプロットすることができる。
【０１８９】
ＤＮＡフラグメントをプロットした後のメンブレンには、（１）アルカリ変性、（２）中和および（３）架橋（クロスリンク）の操作を加えた。
【０１９０】
（１）アルカリ変性：０．５Ｍ　ＮａＯＨ，１．５Ｍ　ＮａＣｌという溶液で飽和した濾紙の上にメンブレンを２分間置いた。
【０１９１】
（２）中和：まず、０．５Ｍ　Ｔｒｉｓ−ＨＣｌ　ｐＨ７．５，１．５Ｍ　ＮａＣｌという溶液で飽和した濾紙の上にメンブレンを５分間置き、続いて２×ＳＳＣで飽和した紙の上にメンブレンを１分間置いた。そしてメンブレンを乾いたペーパータオルの上に置き、約３０分間風乾した。
【０１９２】
（３）架橋：ＵＶクロスリンカー（フナコシ株式会社、東京）を用いて、７０ｎＪ／ｃｍ^２の強さの紫外線による架橋を行った。
【０１９３】
（実施例９：マイクロアレイを用いる、ＥＳ細胞ｍＲＮＡ発現パターンの解析）
ＥＳ細胞におけるｍＲＮＡ発現パターンを解析するために、ＥＳ細胞ｍＲＮＡからプローブを調製して、マイクロアレイ上に固定化した核酸とハイブリダイズさせる。
【０１９４】
（マイクロアレイの調製）
メンブレンフィルターを使用して、以下のとおりマイクロアレイを調製する。
【０１９５】
マクロアレイを、以前に記載のように、市販のグリッディングロボット（ＢｉｏＧｒｉｄ；ＢｉｏＲｏｂｏｔｉｃｓ，Ｃａｍｂｒｉｄｇｅ，ＵＫ）を使用して、Ｈｙｂｏｎｄ　Ｎ＋メンブレン（Ｈｙｂｏｎｄ　Ｎ＋、Ａｍｅｒｓｈａｍ　Ｂｉｏｓｃｉｅｎｃｅｓ，Ｐｉｓｃａｔａｗａｙ，ＮＪ，ＵＳＡ）上にＩＭＡＧＥｃＤＮＡクローン（Ｉ．Ｍ．Ａ．Ｇ．Ｅ＝Ｉ．Ｍ．Ａ．Ｇ．Ｅとはコンソーシアムの名称。Ｕ．Ｓ．Ｄｅｐａｒｔｍｅｎｔ　ｏｆ　Ｅｎｅｒｇｙが後援しているＥＳＴプロジェクトにおいて見出されたｃＤＮＡをデータベース化する団体）由来のＰＣＲ産物の溶液を、スポットすることによって調製した（１０ｎｇ　ＤＮＡ／スポットの量）（Ｎｇｕｙｅｎ，ｃ．ら、Ｇｅｎｏｍｉｃｓ，２９，２０７−２１５、およびＢｅｒｔｕｃｃｉ，Ｆ．ら、Ｏｎｃｏｇｅｎｅ，１８，３９０５−３９１２）。同じ支持体でのマイクロアレイを、Ａｃａｄｅｍｉａ　Ｓｉｎｉｃａの研究者によって開発された固体ピンマイクロスポッティングデバイスを使用して調製した（Ｃｈｅｎ，Ｊ．Ｊ．Ｗ．ら、Ｇｅｎｏｍｉｃｓ，５１−３１３−３２４）。これらの実験に使用したクローンのセットは、例えば、ｈｔｔｐ：／／ｔａｇｃ．ｕｎｉｖ−ｍｒｓ．ｆｒ／ｐｕｂ／Ｃａｎｃｅｒ／に示されるセットである。
【０１９６】
上記で調製した５×４ｍｍ^２のナイロンメンブレン上には、２００個のｃＤＮＡクローンがスポットされる。このように調製したメンブレンに、０．１μｇの総ＲＮＡから作製した^３３Ｐ標識したプローブをハイブリダイズさせる。ハイブリダイゼーション容量は、１００μｌである。ハイブリダイゼーション後、ホスホロスクリーン（高解像度スキャナ）で画像化する。検出限界（ｍＲＮＡ量）は、１／１０，０００であり、検出のための最小サンプル量、０．２×１０^６分子である。
【０１９７】
（ＥＳ細胞ｍＲＮＡからのプローブの調製）
常法を用いて、ＥＳ細胞からｍＲＮＡを調製した後、プローブを調製する。この方法は以下のとおりである。
【０１９８】
１μｌのｄＴ２５（４．１μｇ／μｌ）、０．３μｌのｄＴ１２−１８（０．５μｇ／μｌ）（Ｉｎｖｉｔｒｏｇｅｎ）およびポリＡ　ＲＮＡ（２００ｎｇ）を、ＤＥＰＣ処理したＨ_２Ｏ中に最終容量２．３μｌにまで溶解する。
【０１９９】
この溶液を、７０℃で１０分間インキュベートする。次いで、２．５μｌの５×第１鎖緩衝液（２５０ｍＭ　Ｔｒｉｓ−ＨＣｌ（ｐＨ８．３）、３７５ｍＭ　ＫＣｌ、１５ｍＭ　ＭｇＣｌ_２）、１．２５μｌの０．１Ｍ　ＤＴＴ、１μｌの１０ｍＭ　ｄＡ／ｄＴ／ｄＧおよび０．１μｌの０．１ｍＭ　ｄＣを添加する。得られた混合溶液に、５μｌの［^３３Ｐ］ｄＣＴＰ（３０００Ｃｉ／ｍｍｏｌ）を添加し、４３℃で２分間インキュベートする。次いで、０．５μｌのＳｕｐｅｒＳｃｒｉｐｔＩＩ（Ｉｎｖｉｔｒｏｇｅｎ）を添加し、４３℃（または３７℃）で１時間インキュベートする。次いで、０．５μｌの０．５Ｍ　ＥＤＴＡ、０．５μｌの１％　ＳＤＳおよび１．５μｌの３Ｍ　ＮａＯＨを添加し、６８℃で３０分間インキュベートする。インキュベーション後、室温で１５分間放置する。次いで、５μｌの１Ｍ　Ｔｒｉｓ−ＨＣｌ（ｐＨ７．４）、１．５μｌの２ＮＨＣｌ、２．５μｌの３Ｍ　ＮａＯＡｃおよび５０μｌの１００％　冷ＥｔＯＨを添加し、−２０℃で３０分以上放置する。次いで、４℃で１０分間、１５０００ｒｐｍで遠心分離し、上清を廃棄する。ペレットに、３００μｌの７０％　冷ＥｔＯＨを添加し、４℃で５分間、１５０００ｒｐｍで遠心分離する。上清を廃棄し、ペレットを風乾する。次いで、２５．５μｌの滅菌したｍｉｌｉＱを加えて、ボルテックスする。この溶液の０．５μｌ（１／５０容量）を、液体シンチレーションカウンティングによって放射活性を測定する。次いで、１／２０００ｃｐｍの標識したｔｅｔ−１を添加する。次いで、５μｌのｃｏｔ−１（１０．０μｇ／μｌ）および０．２μｌのポリＡ（１０．０μｇ／μｌ）を添加し、１００℃で５分間インキュベートする。
【０２００】
（ＥＳ細胞ｍＲＮＡプローブとマイクロアレイ上の核酸とのハイブリダイゼーション）
以下のとおり、上述のように調製したマイクロアレイ上に固定化した核酸に対して、上述のように調製したプローブをハイブリダイズさせる。
【０２０１】
メンブレンを、５００μｌのＰｅｒｆｅｃｔ　Ｈｙｂ（東洋紡）中で、６８℃で３０分以上プレハイブリダイゼーションに供する。次いで、２５０μｌの新しいＰｅｒｆｅｃｔ　Ｈｙｂ中で、６８℃で３０分間インキュベートする。上記で調製したプローブを添加し、６８℃で一晩インキュベートする。次いで、２×ＳＳＣ＋１％　ＳＤＳ中で１５分間、２回洗浄し、０．１×ＳＳＣ＋１％　ＳＤＳ中で３０分間、２回洗浄する。メンブレンを乾燥し、高解像度ＩＰに直接露光する。
【０２０２】
（実施例１０；マイクロアレイを用いた解析）
マイクロアレイでの、解析を以下のように行った。
【０２０３】
１）標識ｃＤＮＡの調製
試験対象の動物を、屠殺し、組織を、液体窒素中で凍結する。凍結した組織から、以下の方法を用いて、総ＲＮＡを抽出する。
【０２０４】
総ＲＮＡの抽出方法：グアニジン（ＧＴＣ）溶解／セシウム（ＣｓＣｌ）超遠心分離法（Ｃｈｉｒｇｗｉｎ，Ｊ．Ｊ．ｅｔ　ａｌ．１９７９、Ｂｉｏｃｈｅｍｉｓｔｒｙ　１８，５２９４）に従って、凍結された動物組織より総トータルＲＮＡを抽出した。
【０２０５】
調製したＲＮＡとスーパースクリプトＩＩ逆転写酵素（Ｉｎｖｉｔｒｏｇｅｎ、Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を製造業者のマニュアルに準じて使用して、以下のとおりにｃＤＮＡを合成した：

の反応溶液を調製して、６５℃で、５分間インキュベートして、次に氷上に２〜３分以上放置した。次に、

の反応液を調製して、上記液に加え、４２℃で１時間インキュベートした。次に以下のものを加えた。
【０２０６】
１．０μｌ　　１０％　ＳＤＳ
１．０μｌ　　０．５Ｍ　ＥＤＴＡ
３．０μｌ　　３Ｍ　ＮａＯＨ
これを、６８℃、３０分間インキュベートすることによって、ＲＮＡを分解した。次いでチューブを室温に戻した。
【０２０７】
次に、室温にて以下のものを加えて反応を中和した。
【０２０８】
１０μｌ　１Ｍ　Ｔｒｉｓ−ＨＣｌ　ｐＨ７．５
３μｌ　２Ｎ　ＨＣｌ。
【０２０９】
ｃＤＮＡに取り込まれなかったフリーのヌクレオチド（放射性のものを含む）は、Ｓｅｐｈａｄｅｘ（登録商標）　Ｇ５０（Ａｍｅｒｓｈａｍ　Ｂｉｏｓｃｉｅｎｃｅｓ　Ｃｏｒｐ．　Ｐｉｓｃａｔａｗａｙ，ＮＪ，ＵＳＡ）カラムにより取り除いた。
【０２１０】
２）マイクロアレイ上でのハイブリダイゼーション
マイクロアレイでのハイブリダイゼーションを以下のように行った。
【０２１１】
ハイブリダイゼーションをハイブリダイゼーションオーブン（ＵＶＰ　ＨＢ−１０００，ＵＶＰ，Ｉｎｃ．Ｕｐｌａｎｄ，ＣＡ，ＵＳＡ）を利用して行った。１１．０×７．５ｃｍのナイロン膜に対して、２．０ｍｌのハイブリダイゼーション緩衝液（７５０ｍＭ　ＮａＣｌ；５０ｍＭ　ＮａＨ_２ＰＯ_４；５ｍＭ　ＥＤＴＡ；０．１％Ｆｉｃｏｌｌ；０．１％ＰＶＰ；０．１％　ＢＳＡ；１２０μｇ／ｍｌ　サケ精子ＤＮＡ；１０％デキストランサルフェート；１．０％　ＳＤＳ）と上述プロトコルにより作製したｃＤＮＡプローブを用いて行った。
【０２１２】
ハイブリダイゼーションは、６８℃で２４時間行った。
【０２１３】
ナイロン膜（フィルター）の洗浄は、以下の手順を用いた。
【０２１４】
（１）２×ＳＳＣ、５５℃、３０分間
（２）０．１×ＳＳＣ、０．１％　ＳＤＳ、６５℃、３０分間。
【０２１５】
検出されたハイブリダイゼーションシグナルを、標識したｃＤＮＡの供給源間で比較し、特異的発現パターンを示すｃＤＮＡを同定した。
【０２１６】
（コンピュータによる解析）
オートラジオグラフィーは、ＢＡＳ５０００（富士フィルム、東京）を利用して行い、ドットパターンの解析は、解析ソフトウェア「ＡｒｒａｙＧａｕｇｅ　Ｖｅｒ．１．２」（富士フィルム、東京）を用いて行った。
【０２１７】
（実施例１１；ノックアウト動物の作製）
実施例１０で、脳由来のｃＤＮＡスクリーンで発現し、肝臓由来のｃＤＮＡスクリーンで有意な発現が認められなかったスポットを特異的発現パターンを示すとして選択した。その模式図を図３に示す。特異的発現パターンを示すことが同定されたこのスポットに対応するＥＳ細胞を選択し、常法に従って、Ｃ５７ＢＬ／６マウスの胚盤胞（ｂｌａｓｔｏｃｙｔｅ）にマイクロインジェクションした。そのように処理した胚盤胞を、偽妊娠させたマウスの子宮内に戻した。その結果、キメラ率が非常に高いマウス（微量注入したＥＳ細胞クローン由来の細胞を非常に高い割合（９０％以上）で有する）が６匹（雄性４匹、雌性２匹）生まれた。そのうちの雄性２匹を用いて、正常マウスとの交配を行った。
【０２１８】
染色体中に挿入されたジーントラップベクターを有するＦ_１マウスを選択し、交配させ、ホモ接合型でジーントラップベクターを有するＦ_２マウスを調製する。
【０２１９】
（実施例１２；ノックアウト動物を用いた遺伝子解析）
キメラ率が９０％以上の雄２匹と正常Ｃ５７／ＢＬ６雌マウスを交配したところ、遺伝子（ＧＡＢＡ　Ｃレセプターｒｈｏ３サブユニット遺伝子）がＲＥＴベクターによってトラップ（破壊）された対立遺伝子（アレル）をヘテロに持つマウスが多数生まれた。外部から観察する限り、これらのヘテロマウスには神経学的な異常所見は認められなかった。そこで、これらのヘテロマウス同士を交配することにより、ＧＡＢＡ　Ｃレセプターｒｈｏサブユニット遺伝子がＲＥＴベクターによって破壊された対立遺伝子をホモに持つノックアウト動物を作製した。
【０２２０】
（実施例１３：既知配列を利用したノックアウト動物の作製）
実施例８において配列決定された配列情報を元に、３’ＲＡＣＥ法でＤＮＡフラグメントを合成する代わりに、そのような配列情報を用いてマイクロアレイを作製した。そのような場合、既知の配列情報に基づいてＤＮＡフラグメントを合成する。合成は、核酸自動化合成機を使用する。あるいは、マイクロアレイ上で直接オリゴヌクレオチドを合成することも可能である。
【０２２１】
２）マイクロアレイ上でのハイブリダイゼーション
マイクロアレイでのハイブリダイゼーションを以下のように行った。
【０２２２】
ハイブリダイゼーションをハイブリダイゼーションオーブン（ＵＶＰ　ＨＢ−１０００，ＵＶＰ，Ｉｎｃ．Ｕｐｌａｎｄ，ＣＡ，ＵＳＡ）を利用して行った。１１．０×７．５ｃｍのナイロン膜に対して、２．０ｍｌのハイブリダイゼーション緩衝液（７５０ｍＭ　ＮａＣｌ；５０ｍＭ　ＮａＨ_２ＰＯ_４；５ｍＭ　ＥＤＴＡ；０．１％Ｆｉｃｏｌｌ；０．１％ＰＶＰ；０．１％　ＢＳＡ；１２０μｇ／ｍｌ　サケ精子ＤＮＡ；１０％デキストランサルフェート；１．０％　ＳＤＳ）と上述プロトコルにより作製したｃＤＮＡプローブを用いて行った。
【０２２３】
ハイブリダイゼーションは、６８℃で２４時間行った。
【０２２４】
ナイロン膜（フィルター）の洗浄は、以下の手順を用いた。
【０２２５】
（１）２×ＳＳＣ、５５℃、３０分間
（２）０．１×ＳＳＣ、０．１％　ＳＤＳ、６５℃、３０分間。
【０２２６】
検出されたハイブリダイゼーションシグナルを、標識したｃＤＮＡの供給源間で比較し、特異的発現パターンを示すｃＤＮＡを同定した。
【０２２７】
（コンピュータによる解析）
オートラジオグラフィーは、ＢＡＳ５０００（富士フィルム、東京）を利用して行い、ドットパターンの解析は、解析ソフトウェア「ＡｒｒａｙＧａｕｇｅ　Ｖｅｒ．１．２」（富士フィルム、東京）を用いて行った。
【０２２８】
検出されたハイブリダイゼーションシグナルを、標識したｃＤＮＡの供給源間で比較し、特異的発現パターンを示すｃＤＮＡを同定した。
【０２２９】
以上のように、本発明の好ましい実施形態を用いて本発明を例示してきたが、本発明は、特許請求の範囲によってのみその範囲が解釈されるべきであることが理解される。本明細書において引用した特許、特許出願および文献は、その内容自体が具体的に本明細書に記載されているのと同様にその内容が本明細書に対する参考として援用されるべきであることが理解される。
【０２３０】
【発明の効果】
本発明により、従来に比してはるかに簡便に所望の性質をもつノックアウトマウスを作製することができるようになった。また、従来の方法では同定不可能であった所望の性質をもつ新規遺伝子を同定することができるようになった。
【図面の簡単な説明】
【図１】図１は、本発明で用いたレトロウイルスベクター（ｐＲＥＴジーントラップベクター）の構造を示す。
図１Ａは、パッケージング細胞株中でのｐＲＥＴベクターの構造を示す。ｐＲＥＴベクターの５’ＬＴＲは、野生型配列を有するが、３’ＬＴＲは、Ｕ３部分の転写エンハンサーエレメントを有さない（このベクターは、「自己不活化」レトロウイルスベクターである）。その代わりに、３’ＬＴＲのＵ３部分は、ｌｏｘＰシグナルを有する。
図１Ｂは、感染細胞中でのｐＲＥＴの構造を示す。３’ＬＴＲのヌクレオチド配列は、逆転写および染色体への挿入の過程で、５’ＬＴＲに正確にコピーされる。従って、感染細胞内での５’ＬＴＲもまた、エンハンサーを欠き、そして３’ＬＴＲ内のｌｏｘＰシグナルと同一の配向のｌｏｘＰシグナルを有する。ｐＲＥＴウイルスは、ウイルス転写と逆方向において、以下の３つのエレメントを保有する：（ｉ）強力なスプライスアクセプター（ＳＡ）からなる遺伝子ターミネーターカセット、複数の終止コドン、ＩＲＥＳ配列、ＧＦＰのｃＤＮＡ、および効率的なポリＡシグナル；（ｉｉ）ウイルスの力価決定および陰性選択のための、完全かつ構成性のＨＳＶ　ｔｋカセット；ならびに（ｉｉｉ）スプライスドナー（ＳＤ）およびｍＲＮＡ不安定化シグナルを含むが、ポリＡシグナルを欠く増強したポリＡトラップのための独特のＮＥＯカセット。
図１Ｃは、Ｃｒｅ媒介性切り出し後に染色体に残るエレメントの構造を示す。斜線を引いた長方形は、ジーントラップベクターによって破壊された遺伝子のエクソンを示す。
Ｅｎ（−）：エンハンサー欠失；Ｐ１：ＭＣ１プロモーター；Ｐ２：ＲＮＡポリメラーゼＩＩプロモーター（短縮形態）；Ｘ−ＧＦＰ　ｍＲＮＡ：ジーントラップベクターによって破壊された遺伝子Ｘの５’半分と、ＧＦＰ　ｃＤＮＡとの融合転写物。
【図２】図２は、本発明で用いたＤＮＡマイクロアレイのチップの概略図を示す。
【図３】図３は、本発明で用いたＤＮＡマイクロアレイでの解析の例示を示す。
【図４】図４は、本発明のジーントラップ法で得られたｃＤＮＡを用いたマイクロアレイでの解析のフローの例示を示す図である。
【図５】図５は、本発明で利用されるノックアウト動物の作製方法の例示を示す。
【図６】図６は、本発明の解析において使用されるコンピュータシステムの一例を示す。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for easily producing a knockout animal having desired properties, and a kit, system, embryonic stem cell and microarray for the method. More specifically, the present invention relates to a method for producing a knockout animal using a microarray, and a kit, a system, an embryonic stem cell, and a microarray therefor.
[0002]
[Prior art]
Following the analysis of the human genome, genome decoding has been completed for rice and other plants, and in recent years, analysis of the function of genes has become an increasingly important issue. Conventional gene function analysis has often been performed focusing on individual genes, and in the future, it is required to easily develop gene functions in large quantities.
[0003]
Under such circumstances, in the last decade or so, the analysis of knockout animals via homologous recombination of embryonic stem (ES) cells has become an important means for the purpose of analyzing gene function. Knockout mammals can be prepared using, for example, a positive negative selection method utilizing homologous recombination (Patent Documents 1 to 3, Non-Patent Document 4, Non-Patent Document 5, etc.). An overview of knockout animal production (also referred to as gene targeting) is described, for example, in Non-Patent Documents 6 to 7, and is becoming increasingly popular.
[0004]
In higher organisms, efficient selection of recombinants is performed by, for example, positive selection using a neomycin resistance gene and negative selection using the HSV thymidine kinase gene or diphtheria toxin gene. Selection of homologous recombinants is performed by knockout PCR or Southern blotting. That is, a part of the target gene is replaced with a neomycin resistance gene for positive selection, etc., and a targeting vector having a negative selection HSVTK gene or the like ligated to its end is prepared, and introduced into ES cells by electroporation. And in the presence of ganciclovir, the resulting colonies are isolated and homologous recombinants are selected by PCR or Southern blot.
[0005]
As described above, when a knockout (target gene recombination, gene disruption) mouse having a mutation with a loss of function or a reduced function is produced by destroying an endogenous target gene, the method involves introducing a mutation into only the targeted gene. Therefore, it is useful for analysis of the gene function.
[0006]
After selecting a desired homologous recombinant, the obtained recombinant ES cells are mixed with normal embryos by the scutellum injection method or the assembly chimera method to produce chimeric mice of ES cells and host embryos. In the blastocyst injection method, ES cells are injected into blastocysts with a glass pipette. In the assembly chimera method, a mass of ES cells is adhered to an 8-cell stage embryo from which the zona pellucida has been removed. The blastocyst into which the ES cells have been introduced is transplanted into the uterus of a pseudopregnant surrogate mother to obtain chimeric mice. Since ES cells have totipotency, they can differentiate into all kinds of cells including germ cells in vivo. When a chimeric mouse having ES cell-derived germ cells is crossed with a normal mouse, a mouse having a heterologous ES cell chromosome is obtained. When this mouse is crossed, a knockout mouse having a homologous modified ES cell chromosome is obtained. . To obtain a knockout mouse having a modified chromosome homozygously from the obtained chimeric mouse, a male chimeric mouse and a female wild-type mouse are bred to produce a heterozygous mouse of the F1 generation, and the male and female born Heterozygous mice are crossed to select F2 generation homozygous mice. Whether or not a desired gene mutation has been introduced in each generation of F1 and F2 can be determined using a method commonly used in the art, such as Southern blotting, PCR, and decoding of a base sequence, as in the assay of recombinant ES cells. Can be analyzed.
[0007]
However, the currently used knockout animal production technology has a drawback that since gene disruption occurs simultaneously in all cells constituting an individual, it is not possible to selectively analyze various gene functions.
[0008]
In addition, the production of the current knockout animal requires that after identifying the target gene, the target gene be destroyed from the beginning and knocked out as described above, which requires a great deal of labor and time, and also requires skill. A successful researcher does not always work. Therefore, it is a work that still requires labor-intensive work.
[0009]
Therefore, as a next-generation technology that overcomes the problem that various gene functions cannot be selectively analyzed, a conditional knockout technology that combines cell type-specific expression of Cre recombinase and site-specific recombination of Cre-loxP has been proposed. Attention has been paid. Conditional knockout mice using Cre-loxP were prepared by introducing a neomycin resistance gene into a position that does not inhibit the expression of the target gene, and then introducing a targeting vector into which the loxP sequence was inserted so as to sandwich an exon to be deleted into ES cells. Isolate the homologous recombinant. A chimeric mouse is obtained from the isolated clone, and a genetically modified mouse is produced. Next, when this mouse is crossed with a transgenic mouse that expresses the site-specific recombinant enzyme Cre derived from the P1 phage of Escherichia coli in a tissue-specific manner, the gene is disrupted only in the tissue expressing Cre (here, , Cre specifically recognizes the loxP sequence (34 bp) and causes recombination at the sequence between the two loxP sequences, which is destroyed). Cre can be expressed in adults by mating with a transgenic mouse having a Cre gene linked to an organ-specific promoter, or using a viral vector having a Cre gene.
[0010]
Even with this method, after the target gene is identified, it is necessary to destroy the target gene from scratch and knock it out, which is extremely labor and time consuming, and even skilled researchers will not always succeed. Not exclusively.
[0011]
As a method for analyzing a specific gene, a gene trap (gene trap) method has attracted attention. In the gene trap method, a reporter gene without a promoter is introduced into a cell, and when that gene is accidentally inserted into the genome, the reporter gene is used to isolate a novel gene (trapping). ) Is done. The gene trap method is a method for efficient insertion mutation and identification of unknown genes based on mouse early embryo manipulation, embryonic stem cell culture, and gene targeting by homologous recombination (Non-Patent Document 8). In the gene trap method, introduction of a gene, selection of an insertion mutant, and phenotypic analysis thereof are relatively easy.
[0012]
In the gene trap method, for example, a gene trap vector in which β-geo which is a fusion gene of lacZ and neo is linked between a splicing / acceptor sequence and a polyA addition signal is introduced into ES cells, and selected with G418. Only those clones that accidentally trapped the gene expressed in ES cells are selected.
[0013]
When a chimeric embryo is produced from the clone thus obtained, various X-gal staining patterns are exhibited depending on the expression pattern of the trapped gene. Thus, in the gene trap method, an unknown gene is isolated, its gene expression pattern is analyzed, and the gene is destroyed.
[0014]
However, in the conventional gene trap method, it was necessary to analyze a clone trapped by chance each time, and thus it was not very useful in a project for searching for a gene for a certain purpose (Patent Document 4). Therefore, there is a problem that the gene trap is not very suitable as a tool for systematic research.
[0015]
[Patent Document 1]
US Patent No. 5,464,764
[Patent Document 2]
U.S. Pat. No. 5,487,992
[Patent Document 3]
U.S. Pat. No. 5,627,059
[Patent Document 4]
JP 2001-54337 A
[0016]
[Non-patent document 1]
Gossler, A .; et al. (1989), Science 244, 463-465.
[Non-patent document 2]
Wurst, W.C. et al. (1995), Genetics 139, 889-899.
[Non-Patent Document 3]
Zambroicz, B .; P. et al. (1998), Nature 392, 608-611.
[Non-patent document 4]
Proc. Natl. Acad. Sci. USA, Vol. 86, 8932-8935, 1989
[Non-Patent Document 5]
Nature, Vol. 342, 435-438, 1989
[Non-Patent Document 6]
Edited by Masanori Muramatsu and Masaru Yamamoto, "Experimental Medicine Separate Volume, New Edition, Genetic Engineering Handbook, Third Edition" (1999, published by Yodosha), especially pages 239 to 256
[Non-Patent Document 7]
Shinichi Aizawa (1995) Separate Volume on Experimental Medicine "Gene Targeting-Generating Mutant Mice Using ES Cells"
[Non-Patent Document 8]
Stanford WL. , Et al. , Nature Genetics
2: 756-768 (2001).
[0017]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to develop a system and a method capable of systematically elucidating the functions of a large number of genes at an animal individual level with a minimum of effort and time.
[0018]
[Means to solve the problem]
As a result of the inventor's intensive studies, the above problems have been achieved by combining gene trap analysis with analysis using a microarray, thereby making it possible to reduce the functions of a large number of genes at the animal individual level with minimal effort and time. Was solved by discovering that it could be unraveled unexpectedly. Accordingly, the present invention provides the following.
[0019]
(1) A method for producing a knockout animal, comprising the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) preparing an oligonucleotide corresponding to the gene based on the sequence of the gene trapped in the modified embryonic stem cell;
3) obtaining genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe;
4) selecting a gene having a desired property based on the genetic information;
5) From the modified embryonic stem cells prepared in step 1), select the embryonic stem cells in which the gene having the desired properties has been disrupted, and use the embryonic stem cells in which the genes have been disrupted to produce a knockout animal. Process,
A method comprising:
[0020]
(2) A method for producing a knockout animal, comprising the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) a step of producing a knockout animal from the modified embryonic stem cells prepared in step 1);
3) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
4) obtaining the genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe; and
5) correlating a gene having a desired property selected based on the genetic information with the knockout animal;
A method comprising:
[0021]
(3) The method according to item 1 or 2, wherein the oligonucleotide or a derivative thereof is identifiably labeled.
[0022]
(4) The method according to item 1 or 2, wherein the genetic information is analyzed using a microarray.
[0023]
(5) The method according to item 4, wherein the microarray is a nylon membrane type.
[0024]
(6) The method according to item 1 or 2, wherein the gene trap vector is a retrovirus vector form or an improved poly A trap form.
[0025]
(7) The method according to item 1 or 2, wherein the genetic information is expression level of mRNA, cell or tissue in which mRNA is expressed, expression pattern of mRNA or regulation of mRNA expression.
[0026]
(8) The method according to item 1 or 2, wherein the desired property is to have responsiveness to antibiotic stimulation or to exhibit a significant expression pattern in cells or tissues.
[0027]
(9) The desired properties are that the expression level significantly increases or decreases with the canceration of the cell, that the expression level increases significantly with the induction of cell death, and that the hippocampus controls long-term memory in the brain. 3. The method according to item 1 or 2, which is selected from the group consisting of being specifically expressed in a region.
[0028]
(10) The method according to item 1, wherein the knockout animal is produced by microinjection of embryonic stem cells into blastocysts.
[0029]
(11) The method according to item 1 or 2, wherein the oligonucleotide is a cDNA.
[0030]
(12) A kit for producing a knockout animal,
1) a population of embryonic stem cells whose genes have been gene-trapped; and
2) an oligonucleotide or a derivative thereof corresponding to the gene trapped in each embryonic stem cell in the population of embryonic stem cells,
A kit comprising:
[0031]
(13) The kit according to item 12, wherein the oligonucleotide or the derivative thereof is arranged on a microarray.
[0032]
(14) The kit according to item 12, wherein the embryonic stem cell is provided in a frozen state.
[0033]
(15) The kit according to item 12, wherein the gene is derived from a rat, a mouse, or a hamster.
[0034]
(16) The kit according to item 12, wherein the oligonucleotide or the derivative thereof is about 150 to about 300 nucleotides in length.
[0035]
(17) The kit according to item 13, wherein the microarray contains at least about 1000 oligonucleotides or derivatives thereof.
[0036]
(18) The kit according to item 13, wherein the microarray contains about 1000 to 2000 oligonucleotides or derivatives thereof.
[0037]
(19) The kit according to item 13, wherein the oligonucleotide or the derivative thereof is cDNA.
[0038]
(20) A kit for producing a knockout animal,
1) Gene trap vector;
2) a population of embryonic stem cells; and
3) an oligonucleotide or a derivative thereof corresponding to the gene to be trapped in each embryonic stem cell in the population of embryonic stem cells,
A kit comprising:
[0039]
(21) The kit according to item 20, wherein the oligonucleotide or the derivative thereof is arranged on a microarray.
[0040]
(22) A microarray on which an oligonucleotide or a derivative thereof is arranged, wherein the oligonucleotide or the derivative thereof is a gene trapped in a gene-trapped embryonic stem cell used in a kit for producing a knockout animal. A microarray derived from
[0041]
(23) An oligonucleotide or a derivative thereof corresponding to a gene which has been gene-trapped in the embryonic stem cell and which has been gene-trapped in the embryonic stem cell, is arranged on a microarray used in a kit for producing a knockout animal. Embryonic stem cells.
[0042]
(24) A system for producing a knockout animal,
1) Gene trap vector;
2) a population of embryonic stem cells;
3) an oligonucleotide or a derivative thereof corresponding to a gene to be trapped in each embryonic stem cell in the population of embryonic stem cells;
4) means for analyzing the oligonucleotide or a derivative thereof,
A system comprising:
[0043]
(25) The system according to item 24, wherein the oligonucleotide or the derivative thereof is arranged on a microarray, and the means for analyzing is a means for analyzing the microarray.
[0044]
(26) Further, the following procedure:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) preparing an oligonucleotide corresponding to the gene based on the sequence of the gene trapped in the modified embryonic stem cell;
3) obtaining genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe;
4) selecting a gene having a desired property based on the genetic information;
5) From the modified embryonic stem cells prepared in step 1), select the embryonic stem cells in which the gene having the desired properties has been disrupted, and use the embryonic stem cells in which the genes have been disrupted to produce a knockout animal. Process,
25. The system according to item 24, comprising instructions indicating:
[0045]
(27) Further, the following procedure:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) a step of producing a knockout animal from the modified embryonic stem cells prepared in step 1);
3) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
4) obtaining the genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe; and
5) correlating a gene having a desired property selected based on the genetic information with the knockout animal;
25. The system according to item 24, comprising instructions indicating:
[0046]
(28) A system for producing a knockout animal,
1) a population of gene-trapped embryonic stem cells;
2) an oligonucleotide corresponding to a gene trapped in each embryonic stem cell in the embryonic stem cell population or a derivative thereof; and
3) means for analyzing the oligonucleotide or a derivative thereof;
A system comprising:
[0047]
(29) The system according to item 28, wherein the oligonucleotide or the derivative thereof is arranged on a microarray, and the means for analyzing is a means for analyzing the microarray.
[0048]
(30) A knockout animal produced by the method according to any one of items 1 to 11.
[0049]
(31) A nucleic acid molecule of a gene having desired properties, identified by the method according to any one of items 1 to 11.
[0050]
(32) A polypeptide of a gene having desired properties, identified by the method according to any one of Items 1 to 11.
[0051]
(33) A recording medium for recording genetic information of a gene, obtained by the method according to any one of items 1 to 11.
[0052]
(34) In the method according to item 4, a computer program for causing a computer to execute a method for selecting embryonic stem cells for producing a knockout animal, the method comprising:
1) The above-mentioned genetics on the above-mentioned addresses in the above-mentioned microarray when performing an assay for providing the above-mentioned genetic information using the above-mentioned microarray and the above-mentioned oligonucleotide or its derivative arranged in the above-mentioned microarray Correlating with a signal indicating statistical information; and
2) a step of correlating the address information with the ID of the embryonic stem cell from which the trapped gene is derived;
Wherein selection of genetic information indicative of a desired property indicates the ID of the corresponding embryonic stem cell,
Computer program.
[0053]
(35) In the method according to Item 4, a computer for causing a computer to execute a method of determining which clone or gene in the population corresponds to a spot reflecting a desired property selected on the microarray. A program, wherein the method comprises:
1) selecting the corresponding spot based on the input data of the desired property;
2) selecting a clone or gene correlated to the address of the selected spot;
3) displaying the selected clone or gene;
Including
Computer program.
[0054]
(36) A computer-readable recording medium storing the computer program according to item 34 or 35.
[0055]
(37) The method according to item 4, which is a system for executing a method for selecting embryonic stem cells for producing a knockout animal, wherein the system comprises:
A) computer; and
B) The computer program according to item 34,
Comprising,
system.
[0056]
(38) The method according to item 4, which is a system for judging which clone or gene in the population corresponds to a spot reflecting a desired property selected on the microarray,
A) computer; and
B) The computer program according to item 35,
Comprising,
system.
[0057]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described. It is to be understood that throughout this specification, the use of the singular includes the plural concept unless specifically stated otherwise. It is to be understood that the terms used in the present specification are used in a meaning commonly used in the art unless otherwise specified.
[0058]
(the term)
The definitions of terms used particularly in the present specification are listed below.
[0059]
As used herein, the terms "substrate" and "support" are used interchangeably herein and refer to the material (preferably solid) from which the arrays of the invention are constructed. The material of the substrate may be any solid that has the property of binding to the biomolecule used in the present invention, or that may be derivatized to have such property, either covalently or non-covalently. Materials.
[0060]
As such a material for use as a substrate, any material capable of forming a solid surface can be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (including alloy). , Natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), including but not limited to: The substrate may be formed from multiple layers of different materials. For example, inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used. In addition, polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, polyacrylonitrile, polystyrene, acetal resin Organic materials such as polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, and polysulfone can be used. In the present invention, a membrane used for blotting, such as a nylon membrane, a nitrocellulose membrane, or a PVDF membrane, can also be used. Nylon membranes are preferred. This is because when a nylon membrane is used, the results can be analyzed using a simple analysis system. However, when analyzing a high-density material, it is preferable to use a material having hardness, such as glass.
[0061]
As used herein, the term “chip” or “microchip” refers to a micro integrated circuit that has various functions and becomes a part of a system. In the present specification, the “DNA chip” includes a substrate and DNA, and at least one DNA (for example, a cDNA fragment) is arranged on the substrate. A “DNA chip” is herein included in a “microchip” or simply “chip”. The term “microarray” refers to an array in which one or more biomolecules (eg, oligonucleotides such as cDNA fragments) are arranged on such a chip.
[0062]
The biomolecules (for example, oligonucleotides such as DNA) used in the present invention can use those collected from a living body or can be chemically synthesized by a method known to those skilled in the art. For example, oligonucleotides can be prepared by automated chemical synthesis using any of the DNA synthesizers commercially available from Applied Biosystems or the like. Compositions and methods for the synthesis of automated oligonucleotides are described, for example, in US Patent No. 4,415,732, Caruthers et al. (1983); U.S. Patent No. 4,500,707 and Caruthers (1985); U.S. Patent No. 4,668,777, Caruthers et al. (1987).
[0063]
Although any number of biomolecules (eg, DNA) can be placed on the substrate, typically 10 ⁸ Up to 10 biomolecules, in other embodiments up to 10 ⁷ Up to 10 biomolecules ⁶ Up to 10 biomolecules ⁵ Up to 10 biomolecules ⁴ Up to 10 biomolecules ³ Up to 10 biomolecules, or 10 ² Up to individual biomolecules can be placed. In these cases, the size of the substrate is preferably smaller. In particular, the size of a spot of a biomolecule (eg, DNA) can be as small as the size of a single biomolecule (which can be on the order of 1-2 nm). The minimum substrate area is in some cases determined by the number of biomolecules on the substrate. As used herein, the term “biomolecule” refers to a molecule associated with a living organism. As used herein, the term “organism” refers to a biological organism, and includes, but is not limited to, animals, plants, fungi, viruses, and the like. A biomolecule includes, but is not limited to, a molecule extracted from a living body, and is included in the definition of a biomolecule as long as it is a molecule that can affect a living body. Such biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, including DNA such as cDNA, genomic DNA, RNA such as mRNA), polysaccharides. , Oligosaccharides, lipids, small molecules (eg, hormones, ligands, signal transducers, small organic molecules, etc.), composite molecules thereof, and the like, but are not limited thereto. As used herein, a biomolecule may preferably be DNA or RNA.
[0064]
The terms "protein", "polypeptide", "oligopeptide" and "peptide" as used herein are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acids may be natural or non-natural, and may be modified amino acids. The term can also be assembled into a complex of multiple polypeptide chains. The term also includes naturally or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component). This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids) and other modifications known in the art. Included.
[0065]
As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a polymer of nucleotides of any length. The term also includes "derivative oligonucleotides" or "derivative polynucleotides." The term "derivative oligonucleotide" or "derivative polynucleotide" refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual bond between nucleotides, and is used interchangeably. Specific examples of such an oligonucleotide include 2′-O-methyl-ribonucleotide, a derivative oligonucleotide in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and a phosphodiester bond in an oligonucleotide. Is converted to an N3'-P5 'phosphoramidate bond, a derivative oligonucleotide in which ribose and a phosphodiester bond in the oligonucleotide are converted to a peptide nucleic acid bond, and uracil in the oligonucleotide is C- Derivative oligonucleotide substituted with 5-propynyluracil, derivative oligonucleotide in which uracil in the oligonucleotide is substituted with C-5 thiazoleuracil, cytosine in the oligonucleotide is substituted with C-5 propynylcytosine Derivative oligonucleotide in which cytosine in the oligonucleotide is substituted with phenoxazine-modified cytosine, derivative oligonucleotide in which ribose in DNA is substituted with 2′-O-propyl ribose, and Derivative oligonucleotides in which ribose in the oligonucleotide is substituted with 2'-methoxyethoxyribose are exemplified. The oligonucleotide may be single-stranded or double-stranded, but preferably single-stranded, but the present invention is not limited to single-stranded. Therefore, in the present specification, the “derivative” of an oligonucleotide refers to an oligonucleotide containing a derivative of a nucleotide as described above.
[0066]
As used herein, the term "array" refers to a fixed pattern of fixed objects on a substrate or film, or the patterned substrate itself. Among arrays, those patterned on a small substrate (for example, on a 10 × 10 mm substrate) are referred to as microarrays, but in the present specification, microarrays and arrays are used interchangeably. Therefore, a micropattern that is patterned to be larger than the above-mentioned substrate may be called a microarray. For example, an array consists of a set of desired oligonucleotides that are themselves immobilized on a solid surface or membrane. The array preferably contains at least 10 different oligonucleotides. ² , More preferably at least 10 ³ , And more preferably at least 10 ⁴ , Even more preferably at least 10 ⁵ Including These oligonucleotides are preferably placed on a surface of 125 × 80 mm, more preferably 10 × 10 mm. As a format, a plate size such as a 96-well plate, a 384-well plate, or the like, to a size of a slide glass is contemplated.
[0067]
Typically, arrays are biomolecules (eg, DNA, RNA, protein-RNA fusion molecules, proteins, small organic molecules, etc.) bound to capture nucleic acid sequences that are themselves immobilized on a solid surface or membrane. Be composed. Here, "spots" of biomolecules may be arranged on the array. As used herein, “spot” refers to a certain set of biomolecules. As used herein, “spotting” refers to producing a spot of a certain biomolecule on a certain support. Spotting can be performed by any method, and such methods are well known in the art.
[0068]
As used herein, the term "address" refers to a unique location on a substrate that may be distinguishable from other unique locations. The address is suitable for association with the spot with that address, and may take any shape, such that the entity at every each address can be distinguished (eg, optical) from the entity at another address. The address defining shape may be, for example, a circle, an ellipse, a square, a rectangle, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, when there is no need to distinguish between the two, in the present specification, “address” is used as “address”. “Spot” may be used interchangeably.
[0069]
The size defining each address is, among other things, the size of the substrate, the number of addresses on a particular substrate, the amount of analyte and / or reagents available, the size of the microparticles and any method in which the array is used. Depends on the degree of resolution required. The size can be, for example, in the range of 1-2 nm to several cm, but any size consistent with the application of the array is possible.
[0070]
The spatial arrangement and shape defining the address are designed to suit the particular application in which the microarray is used. The addresses can be closely spaced, widely distributed, or sub-grouped into a desired pattern appropriate for a particular type of analyte.
[0071]
Microarrays (also referred to as DNA microarrays when DNA is arranged) are widely reviewed in (Shujunsha, edited by Cell Engineering, “DNA Microarrays and the Latest PCR Method”). Hereinafter, a DNA array and a gene analysis method using the same will be briefly described.
[0072]
The term “DNA array” or “DNA microarray” refers to a device in which DNAs are arrayed and immobilized on a substrate. A DNA array is classified into a DNA macroarray and a DNA microarray according to the size of a substrate or the density of DNA to be placed. In this specification, unless otherwise specified, the DNA array is used interchangeably with the DNA microarray. . Although the boundary between macro and micro is not strictly determined, generally, a “DNA macro array” refers to a high-density filter in which DNA is spotted on a membrane, and is referred to as a “DNA microarray”. Means DNA on a substrate surface such as glass or silicon. There are a cDNA array, an oligo DNA array, and the like depending on the type to be mounted.
[0073]
Among high-density oligo DNA microarrays, applying photolithography technology for semiconductor integrated circuit manufacturing, synthesizing multiple types of oligo DNA at once on a substrate, comparing them to semiconductor chips, It is called "DNA chip". Those produced using this method include GeneChip® (Affymetrix, CA, USA) and the like (Marshall A et al., (1998) Nat. Biotechnol. 16: 27-31 and Ramsay G et al. (1998) Nat. Biotechnol. 1640-44). Preferably, GeneChip (registered trademark) can be used in gene analysis using a microarray in the present invention. Although the DNA chip is defined as described above in a narrow sense, it may also refer to a DNA array or an entire DNA microarray. Therefore, a chip on which a ready-made DNA such as cDNA is arranged at a high density is also used as a DNA chip. Can be called.
[0074]
Since a DNA microarray is a device in which thousands to tens of thousands or more gene DNAs are arranged on a glass substrate at a high density, the DNA microarray is used to express gene expression by hybridization with cDNA, cRNA or genomic DNA. It has become possible to analyze profiles or gene polymorphisms on a genome scale. By this technique, analysis of the signal transduction system and / or transcriptional control pathway (Fambrough D et al. (1999), Cell 97, 727-741), analysis of the mechanism of tissue repair (Iyer VR et al., (1999), Science 283: 83) -87), mechanism of action of drugs (Marton MJ, (1999), Nat. Med. 4: 1293-1301), extensive analysis of gene expression fluctuations in the process of development and differentiation, genes that fluctuate with disease states Identification, or discovery of new genes involved in signal transduction or transcriptional regulation. Regarding gene polymorphisms, it is possible to analyze a large number of SNPs with one DNA microarray (Cargill M et al., (1999), Nat. Genet. 22: 231-238).
[0075]
As a labeling method in the synthetic DNA microarray, for example, a bifluorescent labeling method is exemplified. In this method, two different mRNA samples are each labeled with different fluorescence, competitive hybridization is performed on the same microarray, both fluorescence is measured, and the difference is detected to detect a difference in gene expression. As the fluorescent dye, for example, Cy5 and Cy3 are most used, but not limited thereto. The advantage of Cy3 and Cy5 is that there is little overlap in fluorescence wavelengths. The bifluorescent labeling method can be used to detect mutations or polymorphisms as well as differences in gene expression.
[0076]
In an assay using a DNA microarray, a fluorescent signal hybridized on the DNA microarray is detected by a fluorescence detector or the like. As such a detector, various detectors are available to date. For example, the Stanford University group has developed an original scanner, which combines a fluorescence microscope and a working stage (see http://cmgm.standford.edu/pbrown). Even with a conventional fluorescence image analyzer for gel, such as FMBIO (Hitachi Software Engineering) or Storm (Molecular Dynamics), a DNA microarray can be read if the spots are not so dense. Other available detectors include ScanArray 4000 and 5000 (General Scanning; scan type (confocal type)), GMS418 Array Scanner (Takara Shuzo; scan type (confocal type)), and Gene Tip Scanner (Nihon Laser Electronics; Scan type (non-confocal type)), Gene Tac 2000 (Genomic Solutions; CCD camera type), and the like.
[0077]
Since the data obtained from microarrays is enormous, data analysis software for managing the correspondence between clones and spots and performing data analysis is important. As such software, software attached to various detection systems can be used (Ermolaeva O et al. (1998) Nat. Genet. 20: 19-23). The format of the database includes, for example, a format called GATC (Genetic analysis technology consortium) proposed by Affymetrix.
[0078]
In another embodiment, a nylon membrane spotted with cDNA can be used as a microarray. The cDNA can be labeled with a label, such as a radiolabel. Such a label can be screened as a mark.
[0079]
As used herein, the term “gene” refers to a factor that defines a genetic trait. Usually they are arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes. As used herein, “gene” may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”.
[0080]
As used herein, an oligonucleotide "corresponding to" a gene refers to an oligonucleotide having a sequence that is substantially identical or complementary to the sequence of that gene. The fact that the sequences are substantially the same means that the correspondence between the gene and its oligonucleotide is 1: 1. Thus, preferably, an oligonucleotide "corresponding to" a gene has a sequence that is completely identical to or completely complementary to the sequence of the gene. The nucleotide length of such "corresponding" oligonucleotides can be any length as long as it is consistent with the purpose of the present invention. Preferably, such an oligonucleotide may be, but is not limited to, the full length of the corresponding gene. Here, the full length of the gene may be the full length of the portion corresponding to the structural gene, the full length of the mRNA sequence, or the full length on the genome.
[0081]
As used herein, “homology” of a gene refers to the degree of identity between two or more gene sequences. Thus, the higher the homology of a given two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When two gene sequences are directly compared, the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% identical, the genes are homologous.
[0082]
In the present specification, the comparison of similarity, identity and homology between amino acid sequences and base sequences is calculated using BLAST, a tool for sequence analysis, using default parameters.
[0083]
In the present invention, oligonucleotides (for example, cDNA) to be arranged on a microarray can be produced by a molecular biological technique using mRNA from a living body, or can be chemically synthesized by a method known to those skilled in the art. For example, a synthesis method using an automated solid phase peptide synthesizer is described by: Stewart, J .; M. et al. (1984). Solid Phase Peptide Synthesis, Pierce Chemical Co. Grant, G .; A. (1992). Synthetic Peptides: A User's Guide, W.C. H. Freeman; Bodanszky, M .; (1993). Principles of Peptide Synthesis, Springer-Verlag; Bodanszky, M .; et al. (1994). The Practice of Peptide Synthesis, Springer-Verlag; Fields, G .; B. (1997). Phase Peptide Synthesis, Academic Press; Pennington, M .; W. et al. (I 994). Peptide Synthesis Protocols, Humana Press; Fields, G .; B. (1997). Solid-Phase PeptideSynthesis, Academic Press. Oligonucleotides can be prepared by automated chemical synthesis using any of the DNA synthesizers commercially available from Applied Biosystems and the like. Compositions and methods for the synthesis of automated oligonucleotides are described, for example, in US Pat. No. 4,415,732, Caruthers et al. (1983); U.S. Patent Nos. 4,500,707 and Caruthers (1985); U.S. Patent No. 4,668,777, Caruthers et al. (1987).
[0084]
As used herein, “expression” of a gene refers to the production of mRNA that is a transcript of the gene or a polypeptide that is a translation product in a target cell, organism, or the like. As used herein, in a preferred embodiment, gene expression refers to expression at the mRNA level.
[0085]
“Expression level” refers to the level at which a polypeptide or mRNA is expressed in a target cell, organism or the like. Examples of the method for measuring the expression level of the polypeptide at the protein level include immunological measurement methods such as ELISA using specific antibodies, RIA, fluorescent antibody, western blotting, and immunohistological staining. . Methods for measuring expression at the mRNA level include, but are not limited to, molecular biological measurement methods such as Northern blotting, dot blotting, and PCR. As used herein, in a preferred embodiment, the expression level or expression level of a gene refers to the expression level or mRNA level. "Change in expression level" refers to an increase or decrease in the expression level of a polypeptide at the protein level or mRNA level, which is evaluated by any appropriate method including the above-described immunological measurement method or molecular biological measurement method. Means that. As used herein, in a preferred embodiment, a change in the expression level or expression level of a gene refers to a change in the expression level or level of mRNA level.
[0086]
As used herein, the term "specifically expresses" a gene means that the gene is expressed at a specific site or stage of an organism at a different (preferably higher) level than at other sites or stages. .
[0087]
In the present specification, the measurement of the expression level of a gene can be performed under various stringency conditions. Such stringency conditions include, but are not limited to, stringent conditions, moderately stringent conditions, and lowly stringent conditions.
[0088]
As used herein, the term "stringent conditions" refers to conditions under which a probe hybridizes to its target sequence but does not hybridize to other sequences, under well-known conditions commonly used in the art. is there. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5 ° C. below the thermal melting point (Tm) for a particular sequence at a defined ionic strength and pH. Typically, stringent conditions include a salt concentration of at least about 0.01-1.0 M Na ion concentration (or other salt) at a pH of 7.0-8.3 and a short temperature. At least about 30 ° C. for the probe (eg, 10-50 nucleotides). Stringent conditions are also achieved with the addition of destabilizing reagents such as formamide. For example, conditions of 5 × SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30 ° C. are suitable for allele-specific probe hybridization.
[0089]
In practicing the present invention, such conditions are preferably that hybridization is performed at 65 ° C. in the presence of 0.7 to 1.0 M NaCl and then 0.1 to 2 times the concentration of SSC (saline). -Sodium citrate solution (the composition of a 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate), and washing the filter at 65 ° C. Hybridization was performed according to Molecular Cloning 2nd ed. , Current Protocols in Molecular Biology, Supplements 1-38, DNA Cloning 1: Core Technologies, A Practical Approach, Second Edition, which can be found in the Second Edition of Experiments, which can be found in Experiments in the Second Edition, which can be found in the Experiments, etc.
[0090]
Specific hybridization refers to hybridization to only specific nucleotide sequences under stringent conditions when the sequences are present in DNA or RNA in a complex mixture. Where T _m Is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (The target sequence is generally present in excess so that at Tm, 50% of the probes are occupied at equilibrium). Perfectly matched probes have a sequence that is completely complementary to a particular target sequence.
[0091]
Test probes, such as those used for expression analysis on arrays, are typically perfectly complementary to a portion (sequence) of the target sequence. The term "mismatch probe" refers to a probe that is deliberately selected such that its sequence is not completely complementary to a particular target sequence. Mismatches can be located anywhere in the mismatch probe, but terminal mismatches are less desirable. This is because terminal mismatches are unlikely to prevent target sequence hybridization. Thus, probes are often designed to have a mismatch located at or near the center of the probe, such that the mismatch is most likely to destabilize the duplex with the target sequence under test hybridization conditions. Is done.
[0092]
Transcription levels can be quantified absolutely or relatively. Absolute quantification is by inclusion of one or more known concentrations of the target nucleic acid, or using a known amount of the target nucleic acid itself, and with reference to the unknown hybridization intensity with the known target nucleic acid (eg, standard (By creating a curve). Alternatively, relative quantification may be achieved by comparison of the hybridization signal between two or more polymorphic forms of a transcript. Such an analysis can be performed using a computer system. As software for performing such an analysis, for example, ArrayGauge Ver. 1.2, ImageGauge Ver. 3.45 (both are Fuji Film Corporation), but are not limited thereto.
[0093]
The techniques used herein are within the skill of the art, unless otherwise indicated, microfluidics, microfabrication, organic chemistry, biochemistry, genetic engineering, molecular biology, unless otherwise indicated. Use well-known and customary techniques in microbiology, genetics and related fields. Such techniques are explained fully, for example, in the references listed below and in other references cited elsewhere herein.
[0094]
For microfabrication, see, for example, Campbell, S.M. A. (1996). The Science and Engineering of Microelectronic Fabrication, Oxford University Press; V. (1996). Microarray Fabrication: a Practical Guide to Semiconductor Processing, Semiconductor Services; Madou, M .; J. (1997). Fundamentals of Microfabrication, CRC15 Press; Rai-Choudhury, P .; (1997). Handbook of Microlithography, Micromachining, & Microfabrication: Microlithography, and the like, the relevant portions of which are incorporated herein by reference.
[0095]
Molecular biology and recombinant DNA technology are described, for example, in Maniatis, T .; et al. (1982). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Ausubel, F.M. M. (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, F .; M. (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Sambrook, J .; et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor; Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, F.C. M. (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Greene Pub. Associates; Ausubel, F .; M. (1995). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates; Innis, M .; A. et al. (1995). PCR Strategies, Academic Press; Ausubel, F .; M. (1999). Short Protocols in Molecular Biology: A Compendium of Methods from Current Current Protocols in Molecular Biology, Wiley, and annual updates; J. et al. (1999). PCR Applications: described in Protocols for Functional Genomics, Academic Press, and the like, the relevant portions of which are incorporated herein by reference.
[0096]
For nucleic acid chemistry such as DNA synthesis technology, see, for example, Gait, M .; J. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; J. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F.C. (1991). Oligonucleotides and Analogues: A Practical Approac, IRL Press; Adams, R.A. L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z .; et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T. (I 996). Bioconjugate Technologies, Academic Press and the like, the relevant portions of which are incorporated herein by reference.
[0097]
Photolithography technology is described in Fordard et al. And utilizes a photoreactive protecting group (see Science, 251, 767 (1991)). This protective group has a function of inhibiting the binding to each base monomer of the same type or another type of base monomer, and no binding reaction of a new base occurs at the base terminal to which this protecting group is bonded. This protecting group can be easily removed by light irradiation. First, an amino group having this protective group is immobilized on the entire surface of the substrate. Next, light irradiation is selectively performed only on the spot to which a desired base is to be bound, using a method similar to the photolithography technique used in a normal semiconductor process. Thereby, only the base in the portion irradiated with the light can introduce the next base by the subsequent binding. Here, a desired base having the same protecting group at the terminal is bound. Then, the shape of the photomask is changed, and another spot is selectively irradiated with light. Thereafter, a base having a protecting group is bonded in the same manner. This process is repeated until a desired base sequence is obtained for each spot, thereby producing a DNA array. Herein, photolithographic techniques may be used.
[0098]
As used herein, "knockout", when referring to a gene, refers to disruption (deficiency) or malfunction of the gene.
[0099]
As used herein, the term “knockout animal” refers to an animal (eg, a mouse) in which a certain gene has been knocked out.
[0100]
As used herein, "animal" may be any animal that can be knocked out. Thus, animals include vertebrates and invertebrates. Animals include mammals (eg, mice, dogs, cats, rats, monkeys, pigs, cows, sheep, rabbits, dolphins, whales, goats, horses, etc.), birds (eg, chickens, quails, etc.), amphibians (eg, , Frogs, etc.), reptiles, insects (eg, Drosophila). Preferably, the animal can be a mammal, more preferably an animal (eg, a mouse) that is easy to make a knockout. In another preferred form, the animal can be an animal (eg, a monkey) that has been found to be suitable as a human model animal. In certain embodiments, the animal can be, but is not limited to, a non-human animal.
[0101]
As used herein, the term “stem cell” refers to a cell that has a self-renewal ability, has a pluripotency, and can regenerate when a tissue is damaged, and includes an embryonic stem (ES) cell, Tissue stem cells (also called tissue-specific stem cells or somatic stem cells) and the like. The stem cells used in the present invention are usually embryonic stem cells, but are not limited thereto. "Embryonic stem cells" refers to pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981, and have been applied to the production of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Therefore, in one preferred embodiment of the present invention, embryonic stem cells can be used as a material for producing a knockout animal, but even if the cells are not embryonic stem cells, they can be used in the present invention as long as the knockout animal can be produced. Can be used.
[0102]
As used herein, the term "retrovirus" refers to a virus that has genetic information in the form of RNA, and that synthesizes DNA from RNA information using reverse transcriptase. Therefore, the term "retrovirus vector" refers to a form in which a retrovirus is used as a carrier (vector) for a gene. Examples of the “retrovirus vector” used in the present invention include, but are not limited to, a retrovirus-type expression vector based on Moloney Leukemia Virus (MMLV), and Murine Stem Cell Virus (MSCV).
[0103]
Preferably, retroviral vectors include, but are not limited to, pGen-, pMSCV, and the like.
[0104]
In the present specification, the “gene trap (method)” refers only to a case where, for example, a reporter gene lacking a promoter is introduced into a target cell and inserted downstream of a promoter activated on a chromosome. This refers to a method for identifying a gene that utilizes reporter activity. Such a gene trap is achieved by introducing a “gene trap vector” into the eukaryotic host chromosome to disrupt the host gene. Since the gene into which the reporter gene is inserted expresses a complex protein with the reporter, the gene can be identified by monitoring the protein. Therefore, the reporter gene is integrated into the original locus as in the case of homologous recombination, and thus a reporter system with complete transcription regulation can be created. By using this technique, a gene that could not be obtained by the technique of isolating a mutant by gene disruption can be identified.
[0105]
In the present specification, the “gene trap vector” is a vector for selecting a vector inserted into a gene by utilizing a phenomenon of undergoing splicing in the process of eukaryotic gene mRNA becoming mature mRNA. Examples of the gene trap vector include (1) a vector containing a coding region of a reporter gene without a promoter and a DNA sequence containing a splice acceptor site, or (2) a coding region of a reporter gene having a promoter and a splice donor site. And (3) a vector containing both the DNA sequences (1) and (2), but not limited thereto. Examples of the gene trap vector shown in (3) include a pRET trap vector (Yasumasa Ishida and Philip Lader, Nuc. Acids Res. 1999, Vol. 27, No. 24, e35). Not limited. This pRET vector is shown in FIG. 1A. The pRET vector can (1) efficiently trap genes that are not expressed in target cells at all, (2) completely destroy the function of the trapped genes, and (3) monitor GFP expression of the trapped genes. (4) It has excellent effects such as being able to cut out a vector once inserted into the genome.
[0106]
The gene trap vector containing the splice acceptor sequence as described above may contain a polyA addition signal, if necessary. A gene trap vector containing a splice donor sequence may optionally include an enhancer region and / or an mRNA destabilizing region. The poly-A addition signal includes, but is not limited to, "AATAAA".
[0107]
Examples of the promoter used in the present invention include, but are not limited to, the MC1 promoter and the RNApol II promoter.
[0108]
Examples of the enhancer used in the present invention include, but are not limited to, polyoma virus enhancer (PYF441).
[0109]
Splice donor sequences used in the present invention include, but are not limited to, the mouse hprt gene exon 8 splice donor.
[0110]
Splice acceptor sequences used in the present invention include, but are not limited to, the exon 3 splice acceptor of the human bcl-2 gene.
[0111]
In the present specification, “genetic information” of a gene refers to information on the state, properties, and the like of the gene. Such genetic information includes the expression level, the timing of expression, the cells that express it, the specificity for a certain stimulus, the mRNA expression level, the cell or tissue in which the mRNA is expressed, the mRNA expression pattern, and the regulation of mRNA expression. But not limited to them. In the present specification, a method for selecting a gene having a desired property from the above-mentioned genetic information can be selected using any method known in the art. Such genetic information can be analyzed using a microarray.
[0112]
As used herein, a “reporter” molecule or “reporter” gene refers to a molecule (eg, a polypeptide) or gene that can be used as an indicator of gene expression in a cell. As such a molecule, a known reporter protein can be used. For example, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS), β-D-galactosidase, luciferase, green fluorescent protein (GFP) Or aequorin.
[0113]
(Description of a preferred embodiment)
In one aspect, the present invention provides a method for producing a knockout animal. This knockout animal production method is achieved by combining the gene trap method with analysis on a DNA array. Therefore, typically, this method of the present invention comprises the following steps: 1) performing a gene trap using embryonic stem cells and a retrovirus vector to generate modified embryonic stem cells; 2) modifying embryonic stem cells A step of preparing (and identifying) an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in each embryonic stem cell in the stem cell; 3) preparing a microarray using the prepared oligonucleotide 4) providing genetic information of the oligonucleotide using the microarray; 5) selecting a gene having a desired property from the provided genetic information; Producing a knockout animal using the embryonic stem cell in which the gene having the desired property has been disrupted.
[0114]
Here, a method of performing a gene trap using embryonic stem cells and a retrovirus vector is performed using a desired material using a technique known in the art. The gene trap is, for example, a reporter gene lacking a promoter (eg, a luciferase gene, a green fluorescent gene, a β-galactosidase gene (lacZ), an alkaline phosphatase gene, a Cre recombinase gene, etc.) is added to a target embryonic stem cell. Reporter activity is only detected when introduced and inserted downstream of a promoter that is activated on the chromosome. The gene trap vector contains, in addition to the reporter gene, a selection marker gene (eg, a neomycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a rescue marker gene (eg, an ampicillin resistance gene + colicin E1 replication origin), and the like. Here, the selectable marker gene is used to select a host containing the vector, and the rescue marker gene is used to rescue the vector (Joyner, AL Ed. Gene Targeting, 2 ^nd edition "(Oxford University Press, 2000). Gene trapping produces modified embryonic stem cells, which are gene-trapped, where they are trapped. The term “is present” means that the endogenous gene is destroyed by the insertion of the trap vector into the genome, and at the same time, the disrupted gene is marked by the vector.
[0115]
In the method of the present invention, a step of preparing (and identifying) an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in each embryonic stem cell in the modified embryonic stem cell is performed. Since the gene into which the above-described reporter gene has been inserted expresses a complex protein with the reporter, it is possible to identify the gene by monitoring the protein. Make it. The preparation of an oligonucleotide having a particular sequence can be performed using techniques well known in the art, for example, see Joyner, A .; L. ed. "Gene Targeting, 2 ^nd edition "(Oxford University Press, 2000). The oligonucleotide can be labeled with fluorescence, radioactivity, etc., if necessary. Such labeling methods are known in the art. It is well known and is described in the references cited herein.
[0116]
In the method of the present invention, a step of preparing a microarray using the oligonucleotide prepared in the above step is performed. Techniques for making microarrays are well known in the art and are described in the references cited herein. The microarray may preferably have a portion for storing information. The arrangement of oligonucleotides on the microarray can be achieved by directly synthesizing oligonucleotides having the same sequence as the cDNA on a substrate (for example, using a photolithography technique), in addition to directly isolating the oligonucleotides. obtain.
[0117]
Preparation of DNA for microarray can be performed using various methods. For example, clones, PCR amplified fragments, synthetic oligonucleotides, and the like can be used as spot DNA for preparing a macroarray used for expression analysis. For example, a fragment amplified by PCR using the above-mentioned cDNA, cloned clone, etc. as a template and a common sequence primer in a vector may be used as spot DNA. If the cloned DNA is not available, prepare a primer set specific to the gene to be analyzed and use genomic DNA or cDNA synthesized from mRNA as a template to perform the same reaction as an array sample. May be used.
[0118]
Examples of such a preparation method include a method including the following steps: 1) Performing 20 to 30 cycles of PCR in a 100 μl reaction system using a 96-well PCR reaction plate; 2) Part of a sample (1-5 μl) is subjected to agarose gel electrophoresis to confirm the amplification state; 3) 130 μl of isopropanol is added and mixed, and left to stand at room temperature for 20-30 minutes; 4) Centrifugation at 3000 rpm for 20 minutes to remove the solution Thereafter, the precipitate is dried; 5) The precipitate is dissolved in 2 × SSC or TE to an appropriate concentration (0.5-1 μg / ml).
[0119]
Target preparation from an RNA sample may also be utilized. For example, RNA extraction methods include destruction of cell walls by denaturants or enzymes and destruction by glass beads. The hot phenol method may be used as a method that can faithfully measure the amount of RNA present in cells. The hot phenol method is well known in the art. For example, in addition to 1) extracting RNA by the hot phenol method, poly (A) RNA may be prepared. This can be followed by the following steps: 2) Labeling with reverse transcriptase (SSII, SuperScript II Reverse Transcriptase, GibcoBRL). The reaction system was 20 μl (10 μg poly (A) RNA, 1 × reaction buffer (supplied with SSII), 25 ng / ml oligo (dT) primer, 0.5 μM dATP, dGTP, dCTP, 0.2 μM dTTP, 0.1 μM dTTP, 0.2 μM dTTP, 0.2 μM dTTP, 0.2 μM dTTP, 0.2 μM dTTP, 0.1 μM dTTP 1 μM FluoroLink dUTP (Amersham-Pharmacia), 10 μM DTT, 100 U RNasin), and RNAs from two kinds of cells are labeled with FluoroLink Cy3-dUTP and FluoroLink Cy5-dUTP, respectively. 3) After heating at 65 ° C for 2 minutes, transfer to 42 ° C, add 200U SSII, and incubate at 42 ° C for 30 to 40 minutes. 4) Add 200U SSII and further incubate at 42 ° C for 30-40 minutes. 5) Add 20 μl Dw, 5 μl 0.5 M EDTA, 10 μl 1 M NaOH and incubate at 65 ° C. for 60 minutes. 6) Add 25 μl 1 M Tris-HCl (pH 7.5) to neutralize. 7) Concentrate the solution to 10-20 μl using microcon30. 8) After adding 200-400 μl TE, concentrate using microcon 30 to 10-20 μl. This operation is repeated 2-3 times to remove unlabeled dNTP. 9) Of the labeled sample, 1 μl is subjected to agarose gel electrophoresis, and the label is confirmed using a gel scanner capable of detecting fluorescence. In this way, the amount of RNA present in the cell can be accurately measured.
[0120]
In the present invention, a step of providing the genetic information of the gene from which the cDNA is derived from the arranged oligonucleotide using the microarray prepared as described above can be performed. Such genetic information includes expression amount, expression time, expression cell, specificity for a certain stimulus, mRNA expression amount, mRNA expression cell or tissue, mRNA expression pattern, mRNA expression regulation, and the like. Responses of certain genes to stimuli (eg, antibiotics, drugs, hormones, vitamins, ligands, etc.) include, but are not limited to. Since the location of the gene's spot on the microarray has already been determined, simply selecting the spot with the desired result will immediately select the embryonic stem cell in which the gene corresponding to that spot has been disrupted (in theory, (In an instant). Therefore, by simply selecting the gene of interest from the results of the microarray analysis, the material for producing a transgenic animal is obtained.
[0121]
As an analysis using such a microarray, all genes were microarrayed, an assay for quantitatively measuring the gene expression level, measurement of changes in the expression level of all genes in an external stimulus or a gene disruption / amplification engineered strain, and the like were obtained. From the database, it is also possible to develop a mechanical learning algorithm that creates an expression control circuit diagram of all the genes of the model organism, a simulator that assumes gene expression control, and the like.
[0122]
Here, a step of selecting the gene having desired properties from the genetic information provided in the above step is performed. Such a selection step can be performed without using a special technique, for example, because it merely selects a spot on the microarray.
[0123]
As described above, at the moment when the spot of interest is selected, the material (embryonic stem cell) of the knockout animal of the gene corresponding to the spot of interest is in hand, and this embryonic stem cell is used for this purpose. By performing a step of preparing a knockout animal in which a gene having a desired property is disrupted, the method of preparing a knockout animal of the present invention is achieved. Techniques for producing a knockout animal from embryonic stem cells are well known in the art, and those skilled in the art will produce a knockout animal by appropriately setting conditions according to the type of the embryonic stem cell and the disrupted gene. can do. Briefly, using the obtained embryonic stem cells, homologous recombination is performed, and embryonic stem cells in which one allele has been modified and disrupted are selected to produce a knockout animal. Here, the embryonic stem cells obtained by the present invention are injected into blastocysts or 8-cell stage embryo cells of a fertilized egg to obtain a chimeric mouse in which embryonic stem cells and embryo-derived cells are mixed. When this chimeric animal is crossed with a normal animal, a heterozygous animal in which all of one allele has been modified and destroyed is produced. By mating the heterozygous animals, homozygous mice can be produced.
[0124]
In a preferred embodiment, the gene trap vector used in the present invention can be a retroviral vector. Such retroviral vectors include, but are not limited to, retrovirus-type expression vectors (pGen-, pMSCV, etc.) based on Moloney Leukemia Virus (MMLV), Murine Stem Cell Virus (MSCV).
[0125]
In a preferred embodiment, the microarray used in the present invention can be a nylon membrane or a glass slide.
[0126]
In a preferred embodiment, the genetic information used in the present invention can be the expression level and expression pattern of mRNA.
[0127]
In a preferred embodiment, the desired properties for use in the present invention include expression upon drug stimulation, expression upon antibiotic stimulation, expression upon stimulation with a signal messenger, expression in disease states, and in very small numbers of cells responsible for specialized functions. May be expression.
[0128]
The knockout animal used in the present invention can be prepared using various methods. For example, such a method includes injecting a genetically modified embryonic stem cell into a blastocyst derived from a normal mouse. Alternatively, a method of producing a chimeric mouse by aggregating it with an early embryo derived from a normal mouse and producing a knockout mouse as a progeny thereof is not limited thereto.
[0129]
In another aspect, the present invention provides a kit for producing a knockout animal. The kit comprises: 1) a gene trapped embryonic stem cell; and 2) a microarray in which oligonucleotides derived from genes trapped in the embryonic stem cell are arranged.
[0130]
Gene-trapped embryonic stem cells can be produced using techniques well known in the art (Joyner, AL ed. "Gene Targeting, 2 ^nd edition "(Oxford University Press, 2000). The specific method of the gene trap is as described in the above description and as exemplified in the examples.
[0131]
Methods for producing a microarray in which oligonucleotides derived from genes trapped in embryonic stem cells are arranged are well known in the art (DNA Microarrays-A Practical Approach, Edited by Mark Schena, Oxford University Press (1999)). reference). A specific method for producing a microarray is described in the above description, and is as exemplified in the examples.
[0132]
Examples of the form of embryonic stem cells included in the kit include, but are not limited to, a frozen state, a powdered state, a form present in a preservation solution, and a form attached to the bottom of a culture vessel filled with a culture solution.
[0133]
The genes trapped in the present invention include, but are not limited to, those derived from mouse embryonic stem cells.
[0134]
Oligonucleotides arranged on the array are preferably those having a length of 500 bases or more, but those having less than 500 bases can also be used. Therefore, preferably, the oligonucleotide has a length of about 150 to about 800, more preferably about 300 to about 600 nucleotides. Also, those having a length of about 150 to 300 nucleotides may be preferable.
[0135]
In the microarray used in the present invention, spots corresponding to a desired number of genes can be arranged. For example, any number of spots can be placed on the substrate, but typically ⁸ Up to the spot, in another embodiment 10 ⁷ 10 to the spot ⁶ 10 to the spot ⁵ 10 to the spot ⁴ 10 to the spot ³ Up to the spot or 10 ² Spots to spots can be located. Therefore, when one cDNA is placed in one spot, 10 ⁸ Up to 10 cDNAs in another embodiment. ⁷ Up to 10 cDNAs ⁶ Up to 10 biomolecules ⁵ Up to 10 biomolecules ⁴ Up to 10 cDNAs ³ Up to 10 cDNAs or 10 ² CDNAs up to a number of biomolecules can be arranged. In these cases, smaller substrate sizes and smaller numbers are appropriate. In particular, the spot size may be as small as the size of a single cDNA (this may be on the order of 1-2 nm). The minimum substrate area is in some cases determined by the number of spots on the substrate.
[0136]
In another aspect, the present invention provides a microarray on which oligonucleotides are arranged. Here, the oligonucleotide is derived from a gene trapped in a gene-trapped embryonic stem cell used in a kit for producing a knockout animal. Methods for producing such oligonucleotides are as described herein.
[0137]
In another aspect, the invention provides a gene-trapped embryonic stem cell. The present invention is characterized in that the cDNA sequence of a gene that has been gene-trapped in such embryonic stem cells is arranged on a microarray used in a kit for producing a knockout animal. Methods for making such embryonic stem cells are well known in the art and are described herein.
[0138]
The present invention provides a system for producing a knockout animal. The system comprises: 1) a gene trapped embryonic stem cell; 2) an oligonucleotide or a derivative thereof of a gene trapped in the embryonic stem cell; 3) a means for analyzing the oligonucleotide or a derivative thereof. The oligonucleotide or derivative thereof can be arranged on a microarray. The analyzing means may be a means for analyzing the microarray.
[0139]
The method of producing the gene-trapped embryonic stem cells used in such a system is as described herein. The method of making the microarray used in such a system is also as described herein.
[0140]
As a means for analyzing the microarray, various known means can be used. Such means include computer systems. Specific examples of such analysis means include, but are not limited to, GMS418, GMS428 (Affymetrix), Scanarray Express (GSI Lumonics), and GenePix 4000B (Axon). Examples of the analysis software used in this case include Imagegene for GMS, Quantarray for Scanarray Express, and GenePix Pro for Genepix, but a modified version of the software may also be used. When using a nylon membrane as a microarray, radioisotopes ( ³³ P)). In this case, the above-mentioned dedicated analyzer is not particularly necessary, and a highly versatile image analyzer BAS 2500 or BAS 5000 (both Fuji Film) and analysis software ArrayGauge Ver. The data can be analyzed using 1.2 (Fuji Film). The principles of such analysis are well known and are detailed in DNA Microarrays-A Practical Approach, Edited by Mark Schena, Oxford University Press (1999).
[0141]
The system of the present invention may be accompanied by instructions describing how to use the system. The instructions will, where necessary, be prepared in accordance with the format prescribed by the competent authority of the country in which the invention is implemented, and will specify that they have been approved by that competent authority as required. The instruction sheet is a so-called manual, and is usually provided in a paper medium, but is not limited thereto. For example, the instruction sheet may be provided in a form such as an electronic medium (for example, a homepage provided on the Internet, an e-mail).
[0142]
Here, such instructions include the following procedures: 1) performing a gene trap using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells; Preparing an oligonucleotide corresponding to the trapped gene based on the sequence of the trapped gene in the stem cell; 3) using the prepared oligonucleotide or a derivative thereof as a probe to obtain the genetic information of the trapped gene. Obtaining the gene; 4) selecting a gene having a desired property based on the genetic information; and 5) disrupting the gene having the desired property from the modified embryonic stem cells prepared in the step 1). A step of selecting the obtained embryonic stem cells and producing a knockout animal using the embryonic stem cells in which the gene has been disrupted may be shown. These specific steps also include: 1) performing a gene trap using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells; 2) modifying the embryonic stem cell prepared in step 1) A step of preparing a knockout animal from stem cells; 3) a step of preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cells; 4) a step of preparing the prepared oligonucleotide or a derivative thereof. Obtaining genetic information of the trapped gene by using it as a probe; and 5) correlating the knockout animal with a gene having a desired property selected based on the genetic information. Is also good. Although these steps may be described to the extent that those skilled in the art can read and use them, since they may be used by those who do not have knowledge, this instruction includes specific and detailed protocols. It may be. In addition, the instruction does not need to describe all the steps described above, and may include at least one step. Preferably, two or more, three or more, four or more, more preferably all five steps may be described. Further, matters other than these five steps may be described in this instruction sheet.
[0143]
Each of these steps may be specifically described in the protocol in the following protocol format.
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells:
Introduction of a gene trap vector into embryonic stem cells (by retroviral infection); selection with a selectable marker such as G418; picking up a selectable marker-resistant embryonic stem cell colony such as G418; and selectable marker-resistant embryonic such as G418 Stem cell freezing and FACS analysis of GFP;
2) a step of preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell:
RNA extraction from G418-resistant embryonic stem cells; synthesis of cDNA by reverse transcription of the extracted RNA; amplification of DNA fragment of trapped gene by PCR;
3) a step of obtaining the genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe (for example, a step of preparing a microarray using the prepared oligonucleotide and trapping using the microarray) Step of obtaining genetic information of the selected gene):
Purification and further amplification of DNA fragments using gene-specific primers, and spotting of DNA on a support (nylon membrane, slide glass, etc.); reverse transcription of mRNA prepared from several different samples (cells or tissues) Hybridization with the DNA on the prepared microarray, washing and autoradiography of the microarray, and analyzing the results by software;
4) Step of selecting a gene having a desired property based on the genetic information:
Select the desired properties to be studied;
5) A step of selecting, from the modified embryonic stem cells, an embryonic stem cell in which the gene having the desired property is disrupted, and producing a knockout animal using the embryonic stem cell in which the gene is disrupted:
Microinjection of gene-disrupted embryonic stem cells into blastocysts; production of chimeric mice; production of heterozygous mice by crossing chimeric mice with wild-type mice; The desired knockout mouse is selected by the production of both alleles derived from the mutant and its phenotypic analysis.
[0144]
In one aspect, the present invention provides a knockout animal (eg, a mouse) produced by the method of the present invention. Such knockout animals have not been able to be made quickly with conventional techniques.
[0145]
In one aspect, the present invention provides a nucleic acid molecule of a gene having desired properties, identified by the method of the present invention. According to the method of the present invention, it is possible to analyze the function of an unknown gene that could not be performed conventionally. Gene information thus analyzed is information that cannot be obtained by a conventional method, and a nucleic acid molecule having a sequence of a gene having such information is a nucleic acid molecule that cannot be obtained by a conventional technique. It is.
[0146]
In another aspect, the present invention provides a polypeptide of a gene having a desired property, identified by the method of the present invention. As described above, a polypeptide having a sequence of a gene having information that cannot be obtained by the conventional technology is a polypeptide that cannot be obtained by the conventional technology.
[0147]
The present invention provides a recording medium on which genetic information of a gene obtained by the method of the present invention is recorded. The recording medium may be a flexible disk, MO, CD-R, CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, memory card, etc., but is not limited thereto. .
[0148]
The present invention provides a computer program for causing a computer to execute a method of selecting embryonic stem cells for producing a knockout animal for use in the method of the present invention. The method executed by this program includes: 1) the address information of the cDNA arranged in the microarray and the address in the microarray when an assay for providing the genetic information is performed using the microarray. Correlating the signal indicating the genetic information above with 2) correlating the address information with the ID of the embryonic stem cell from which the trapped gene is derived, When genetic information indicating a desired property is selected, the ID of the corresponding embryonic stem cell is indicated.
[0149]
In another aspect, the present invention provides a computer-readable recording medium storing a computer program for causing a computer to execute a method for selecting embryonic stem cells for producing a knockout animal in the method of the present invention. I do. Here, the method executed by the computer program stored in the recording medium includes: 1) address information of the cDNA arranged in the microarray, and an assay for providing the genetic information using the microarray. Correlating the signal indicating the genetic information on the address in the microarray when performed, and 2) correlating the address information with the ID of the embryonic stem cell from which the trapped gene is derived Selecting genetic information indicative of a desired property indicates the ID of the corresponding embryonic stem cell. Here, the recording medium may be a flexible disk, MO, CD-R, CD-RW, CD-ROM, DVD-RAM, DVD-R, DVD-RW, DVD + RW, DVD-ROM, memory card, or the like. It is not limited to. Further, the recording medium may be a hard disk. Thus, an application server provider may provide such a program.
[0150]
In another aspect, the present invention provides a system for performing the method of the present invention for selecting a embryonic stem cell for producing a knockout animal. Such a system comprises: A) a computer; and B) a computer program that causes the computer to perform a method of selecting embryonic stem cells for producing a knockout animal. Here, the method includes: 1) address information of the cDNA arranged in the microarray, and information on the address in the microarray when an assay for providing the genetic information is performed using the microarray. Correlating with the signal indicating the genetic information, and 2) correlating the address information with the ID of the embryonic stem cell from which the trapped gene is derived, wherein the desired When the genetic information indicating the property is selected, the ID of the corresponding embryonic stem cell is indicated. These systems may be at the same location or at different locations. Thus, media at a distance, such as an application server provider, may provide such a program.
[0151]
In another aspect, the present invention provides the method for producing a knockout animal of the present invention, wherein a method for determining which clone or gene in the population corresponds to a spot reflecting a desired property selected on a microarray is included in a computer. Provide a computer program to be executed. Here, the method includes: 1) selecting a corresponding spot based on the input data of the desired property; 2) selecting a clone or gene correlated with the address of the selected spot. 3) displaying the selected clone or gene. These systems may be at the same location or at different locations. Thus, media at a distance, such as an application server provider, may provide such a program.
[0152]
Next, an execution method of the computer according to the present invention will be described with reference to FIG. FIG. 6 shows a configuration example of a computer 500 that executes the processing of the method for selecting embryonic stem cells for producing a knockout animal of the present invention.
[0153]
The computer 500 includes an input unit 501, a CPU 502, an output unit 503, a memory 504, and a bus 505. The input unit 501, the CPU 502, the output unit 503, and the memory 504 are mutually connected by a bus 505. The input unit 501 and the output unit 503 are connected to an input / output device 506.
[0154]
Hereinafter, an outline of processing of a method of selecting embryonic stem cells for producing a knockout animal, which is performed by the computer 500, will be described.
[0155]
A program for executing a method for selecting embryonic stem cells for producing a knockout animal (hereinafter, a selection program) is stored in the memory 502, for example. Alternatively, the selection program can be recorded on any type of recording medium such as a flexible disk, an MO, a CD-ROM, and a DVD-ROM. Alternatively, it may be stored in the application server. The selection program recorded on such a recording medium is loaded into the memory 504 of the computer 500 via the input / output device 506 (for example, a disk drive, a network (for example, the Internet)). When the CPU 502 executes the selection program, the computer 500 functions as an apparatus that executes the processing of the method for selecting embryonic stem cells for producing a knockout animal of the present invention.
[0156]
Through the input unit 501, address information, signal information, information on desired properties, ID information of embryonic stem cells, and sequence information as necessary are input.
[0157]
The CPU 502 correlates the address information with the signal strength data based on the information input from the input unit 501, and stores the correlation data in the memory 504. Next, the CPU 502 correlates the address information with the ID of the embryonic stem cell. Thereafter, the CPU 502 can store such information in the memory 504. The CPU 502 selects an appropriate signal strength based on information on a desired property. The ID of the corresponding embryonic stem cell is calculated based on the address information having the selected signal intensity data. This information can also be stored in memory 504.
[0158]
After that, the output unit 503 outputs the ID data of the embryonic stem cell selected by the CPU 502 and the like. The output data can be output from the input / output device 506.
[0159]
A method for determining which clone or gene in the population corresponds to a spot reflecting a desired property selected on the microarray can also be performed in the same manner as described above using the system of FIG.
[0160]
The methods, kits, programs, systems, etc., of the present invention can be used, for example, in diagnosis, forensics, identification of novel genes, drug discovery (drug screening) and development, molecular biological analysis (eg, array-based nucleotide sequence analysis and Based gene sequence analysis), analysis of protein properties and functions, pharmacogenomics, proteomics, environmental research and further biological, medical, pharmaceutical, agricultural, physical and chemical analyses.
[0161]
The references cited herein, such as scientific literature, patents, patent applications, and the like, are hereby incorporated by reference in their entirety to the same extent as if each were specifically described.
[0162]
The present invention has been described above by showing preferred embodiments for easy understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only, and are not provided for limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but only by the claims.
[0163]
【Example】
In the examples described below, unless otherwise specified, reagents and instruments from Nacalai Tesque, Inc. (Kyoto), Wako Pure Chemical Industries, Ltd. (Osaka), and Sigma (St. Louis, MO, USA) were used. .
[0164]
Example 1 Preparation of Virus by Transfection of Plat-E Virus Packaging Cell Line
1) Culture of Plat-E cells
Plat-E cells (see Morita, S. et al. (2000) Gene Therapy 7, 1063-1066) were placed in Dulbecco's Modified Essential Medium (DMEM) containing 10% fetal bovine serum (FBS) at 10 μg / ml. Blasticidine, 1 μg / ml puromycin, 1 × penicillin (100 U / ml), 1 × streptomycin (100 μg / ml), humidified at 37 ° C., 5% CO 2 ₂ Culture in the presence. Subculture 1/4 to 1/10 volume every 3 days.
[0165]
2) Transfection of Plat-E cells
3 × 10 on 10cm plate ⁶ Seed Plat-E cells. Twenty-four hours after seeding, FuGENE6 reagent (Roche Diagnostics, Mannheim, Germany) is used for transfection as follows according to the manufacturer's instructions.
[0166]
Dispense 100 μl of DMEM into a 1.5 ml Eppendorf tube. Add 9 μl of FuGENE6 reagent directly to the medium. To this mixed solution, 3 μg of a DNA solution (pRET trap vector (Yasumasa Ishida and Philip Lader, Nuc. Acids Res. 1999, Vol. 27, No. 24, e35), FIG. 1A) was added and mixed gently. Incubate at room temperature for 15 minutes. This mixture is added to the cell culture medium. Virus-producing cells were humidified at 37 ° C., 5% CO 2. ₂ Culture in the presence.
[0167]
Forty-eight hours after transfection, the supernatant was collected (10 ml per 10 cm plate) using a 10 ml syringe and filtered through a 0.45 μm membrane (HTuffryn, Pall Gelman Laboratory, Ann Arbor, MI, USA). Remove virus-producing cells and cell debris.Use filtered virus preparations for direct infection or store at -80 ° C.
[0168]
(Example 2: Infection of ES cells using prepared virus)
STO medium (Dulbecco's modified essential medium (DMEM) containing 7% fetal bovine serum (FBS) (high glucose, 4.5 g / L) was supplemented with 2 mM L-glutamine, 50 U / ml penicillin, and 50 μg / ml streptomycin. Supplemented medium) and ES medium (Dulbecco's Modified Essential Medium (DMEM) containing 15% fetal bovine serum (FBS) (high glucose, 4.5 g / L)), 100 μM non-essential amino acids, 2 mM L-glutamine, A medium containing 100 μM 2-mercaptoethanol (2-ME), 50 U / ml penicillin, and 50 μg / ml streptomycin) is used.
[0169]
1) Day 0: Mitomycin C treatment-Preparation of STO plate (M-STO plate)
Before thawing M-STO feeder cells (McMahon, AP and Bradley, A. (1990) Cell 62, 1073-1085), the inner surface of a 10 cm plate is coated with 0.1% gelatin. A vial of M-STO cells (mitotically inactivated mouse embryonic fibroblast cell line (M-STO feeder cells)) is quickly thawed and transferred to a 15 ml tube, to which is added about 10 ml of STO medium. The cells are harvested by centrifugation for 5 minutes at 800 rpm at 4 ° C. After removing the supernatant, the cells are gently suspended in STO medium. Gently plate on turbid and gelatin-treated plates. ₂ Incubate at 37 ° C in the presence.
[0170]
2) Day 1; Lysis of ES cells
The day after plating, the ES cells (RF8 strain, Melner, VL et al. (1996) PNAS 93, 14041-14046) are rapidly lysed at 37 ° C. The cell suspension in the vial is added immediately to the ES medium. After centrifugation at 800 rpm for 5 minutes, the supernatant is removed, and then resuspended in ES medium. Remove STO medium from plate of M-STO cells and add ES medium. Add the ES cell suspension to the plate.
[0171]
3) Day 2-4; ES cell division
The medium is removed from the ES cell plate and the attached cells are washed with PBS. After removing the PBS, add 1 ml of a 0.25% trypsin / EDTA solution and incubate the plate at 37 ° C. for 10-15 minutes. After adding ES medium to neutralize trypsin, pipetting is performed to disperse the released ES cells into single cells. Add ES cell suspension to M-STO plate. Split cells before they reach confluence. At this time, it is important that the cells are not proliferated excessively until they reach confluence, and that when the cells are split, the density is not too low.
[0172]
4) Day 5; infection
Wash plate with 5 ml PBS. One ml of 0.25% trypsin / EDTA solution is added and the plate is incubated at 37 ° C for 15 minutes. ₂ Incubate in incubator. Add 9 ml of ES medium and pipette to break the cells into a single cell suspension. Determine the number of ES cells. Centrifuge at 800 rpm for 5 minutes at 4 ° C. to collect cells. In 15 ml of retroviral supernatant containing 5-10 μg / ml polybrene, 2 × 10 ⁶ The ES cells are suspended. CO ₂ Incubate at 35-37 ° C. for 2 hours in incubator. Centrifuge at 800 rpm for 5 minutes at 4 ° C. to collect cells. The supernatant is removed and the cells are suspended in 20 ml of ES cells. Add 5 ml of cell suspension to a 10 cm plate containing M-STO feeder cells.
[0173]
5) Days 6-13 (or 16); selection at G418
After 32-48 hours of incubation, ES cells medium containing 250 μg / ml Geneticin (Geneticin; G418) is added to all plates. Medium is changed daily during drug selection for 7-10 days until good colonies can be isolated.
[0174]
(Example 3: Isolation of ES cell colonies)
1) Day 0
Prepare 24-well plates of M-STO feeder cells 1-2 days before colony isolation. In this case, the concentration for plating the M-STO feeder cells was 4 × 10 ⁴ Cells / well.
[0175]
2) Day 1
20 μl of a 0.25% trypsin / EDTA solution is added to each well to prepare a 96-well ぽ u rate. The ES medium is removed from the plate containing the ES colony. After washing the plate twice with PBS and removing the PBS, 5 ml of PBS is added to cover the surface of the plate. Colonies are isolated from the washed plates and transferred to a trypsin / EDTA solution in 96 wells. The 96-well plate is incubated for about 15 minutes at ₂ Incubate in incubator. Add 180 μl ES medium per well. Pipette 20 times to break up cells. Transfer each ES cell clone to a numbered 24-well plate containing M-STO feeder cells. Change the ES medium daily.
[0176]
(Example 4: Freezing of ES cells in a 24-well plate)
The medium is removed from the wells and washed with PBS. Add 100 μl of 0.25% trypsin / EDTA solution to each well. Incubate at 37 ° C for 5-10 minutes. Add 700 μl ES medium to each well. The medium in each well is mixed 15-20 times using a P-1000 pipette (Gilson, Inc. (Middleton, WI, USA)) to prepare a single cell suspension.
[0177]
1) Preparation of frozen cells for storage
Transfer 500 μl of each cell suspension to a freeze-vial (cyto-vial; IWAKI Cryogenic Vial, Asahi Techno Glass, Tokyo) containing 500 μl of 2 × freezing medium (DMEM 60%; FCS 20%; DMSO 20%). . Transfer the freezing vial into liquid nitrogen or into a -80 ° C freezer.
[0178]
2) Preparation of frozen cells for FACS analysis
Add 900 μl of fresh ES medium to remaining ES cells in each well and mix gently. Transfer 300 μl of the cell suspension to a 1.5 ml eppendorf tube and centrifuge at 3000 rpm for 5 minutes. Remove the supernatant and suspend in 500 μl PBS.
[0179]
3) Preparation of frozen cells for preparing total RNA
Transfer the remaining cell suspension (approximately 800-900 μl) to a 1.5 ml eppendorf tube and centrifuge at 3000 rpm for 5 minutes. The medium is removed and 500 μl of Sepasol reagent (Nacalai Tesque, Inc., Kyoto) is added. Mix immediately by vortexing and store at -80 ° C.
[0180]
(Example 5: RNA extraction)
Total RNA corresponding to ES cells in each well is extracted using Sepasol reagent (Nacalai Tesque, Inc., Kyoto) according to the manufacturer's instructions.
[0181]
(Example 6; cDNA synthesis)
1) Reverse transcription
CDNA is synthesized using Moloney MLV reverse transcriptase (Invitrogen, Carlsbad, CA, USA) as follows:

And incubate at 65 ° C. for 5 minutes, then leave on ice for 3 minutes or more. next,

And incubate at 37 ° C. for 50 minutes, then at 72 ° C. for 15 minutes. Add 80 μl of 10 mM Tris-HCl (pH 8.0) and store at −20 ° C.
[0182]
(Example 7; 3′RACE)
Using the cDNA prepared from the total RNA corresponding to each well, the 3 'end is prepared by the 3' RACE method (Ohara, O. et al. (1989) PNAS 86, 5673-5677).
[0183]
The following reaction is carried out using an Advantage-GC2 polymerase mix (BD Biosciences Clonetech, Palo Alto, CA, USA) as a polymerase used in the 3′RACE method.
[0184]
1) First RACE reaction

Prepare a reaction solution of the following cycle:

And store the reaction at 4 ° C.
[0185]
2) Second RACE reaction

Prepare a reaction solution of the following cycle:

And store the reaction at 4 ° C.
[0186]
(Example 8: Preparation of microarray)
The nucleotide sequence of the DNA fragment prepared by the 3′RACE method in Example 7 was determined by a direct sequencing method (Kusukakawa, N. et al. 1990, BioTechniques 9: 66-72). NEO3.5 primers were used for direct sequencing. Based on this sequence information, a primer (upstream, ie, antisense) specific to each gene is synthesized. Each gene fragment was re-amplified by PCR using NEO3.5 primer and newly synthesized gene-specific primer. This re-amplified DNA fragment was used for immobilization on a microarray.
[0187]
The absorbance (OD260) of the re-amplified DNA fragment was measured, and the concentration of each fragment was adjusted to 0.5-1.5 pmol / μl in consideration of the length of each fragment. Then, each DNA fragment was transferred to a 96-well plate.
[0188]
For the preparation of the microarray, a multi-pin plotter 96 (AB-6690NM, Ato Corporation, Tokyo) and a Biodyne B nylon membrane (Pall Gelman Laboratory, Ann Arbor, MI, USA) were used. 864 DNA fragments can be plotted on a 11.0 x 7.5 cm membrane.
[0189]
After the DNA fragments were plotted, the membrane was subjected to the operations of (1) alkali denaturation, (2) neutralization, and (3) cross-linking.
[0190]
(1) Alkali denaturation: The membrane was placed on a filter paper saturated with a solution of 0.5 M NaOH and 1.5 M NaCl for 2 minutes.
[0191]
(2) Neutralization: First, place the membrane on a filter paper saturated with a solution of 0.5 M Tris-HCl pH 7.5, 1.5 M NaCl for 5 minutes, and then put the membrane on a paper saturated with 2 × SSC. For 1 minute. Then, the membrane was placed on a dry paper towel and air-dried for about 30 minutes.
[0192]
(3) Crosslinking: 70 nJ / cm using a UV crosslinker (Funakoshi, Tokyo) ² The cross-linking was carried out by ultraviolet light having a strength of.
[0193]
(Example 9: Analysis of ES cell mRNA expression pattern using microarray)
In order to analyze the mRNA expression pattern in ES cells, a probe is prepared from ES cell mRNA and hybridized with a nucleic acid immobilized on a microarray.
[0194]
(Preparation of microarray)
Using a membrane filter, prepare a microarray as follows.
[0195]
Macroarrays were prepared using a commercially available gridding robot (BioGrid; BioRobotics, Cambridge, UK) as described previously, using a Hybrid N + membrane (Hybond N +, Amersham Biosciences, Piscata, USA, cDNA on Nike, USA, cDNA, USA, USA). (IMAGE = IMAGE is the name of the consortium. A database of cDNAs found in the EST project sponsored by the US Department of Energy) A solution of the PCR product from (Group) was prepared by spotting (10 ng DNA / spot amount) (Nguyen, c., Et al., Genomics, 29, 207-215, and Bertuc). i, F., et al., Oncogene, 18,3905-3912). Microarrays on the same support were prepared using a solid pin microspotting device developed by researchers at Academia Sinica (Chen, JJW et al., Genomics, 51-313-324). The set of clones used in these experiments is, for example, http: // tagc. univ-mrs. It is a set shown in fr / pub / Cancer /.
[0196]
5 × 4mm prepared above ² 200 cDNA clones are spotted on the nylon membrane. The membrane thus prepared was prepared from 0.1 μg of total RNA. ³³ The P-labeled probe is hybridized. The hybridization volume is 100 μl. After hybridization, images are formed with a phosphor screen (high-resolution scanner). The detection limit (mRNA amount) is 1 / 10,000, and the minimum sample amount for detection, 0.2 × 10 ⁶ Is a molecule.
[0197]
(Preparation of probe from ES cell mRNA)
After preparing mRNA from ES cells using a conventional method, a probe is prepared. The method is as follows.
[0198]
1 μl of dT25 (4.1 μg / μl), 0.3 μl of dT12-18 (0.5 μg / μl) (Invitrogen) and poly A RNA (200 ng) were treated with DEPC-treated H ₂ Dissolve in O to a final volume of 2.3 μl.
[0199]
This solution is incubated at 70 ° C. for 10 minutes. Then, 2.5 μl of 5 × first strand buffer (250 mM Tris-HCl (pH 8.3), 375 mM KCl, 15 mM MgCl 2 ₂ ), 1.25 μl of 0.1 M DTT, 1 μl of 10 mM dA / dT / dG and 0.1 μl of 0.1 mM dC are added. 5 μl [ ³³ Add P] dCTP (3000 Ci / mmol) and incubate at 43 ° C for 2 minutes. Then, 0.5 μl of SuperScript II (Invitrogen) is added and incubated at 43 ° C. (or 37 ° C.) for 1 hour. Then, 0.5 μl of 0.5 M EDTA, 0.5 μl of 1% SDS and 1.5 μl of 3 M NaOH are added and incubated at 68 ° C. for 30 minutes. After incubation, leave at room temperature for 15 minutes. Then, 5 μl of 1 M Tris-HCl (pH 7.4), 1.5 μl of 2N HCl, 2.5 μl of 3 M NaOAc and 50 μl of 100% cold EtOH are added and left at −20 ° C. for 30 minutes or more. Then centrifuge at 15000 rpm for 10 minutes at 4 ° C. and discard the supernatant. Add 300 μl of 70% cold EtOH to the pellet and centrifuge at 15000 rpm for 5 minutes at 4 ° C. Discard the supernatant and air-dry the pellet. Then add 25.5 μl of sterile miliQ and vortex. 0.5 μl (1/50 volume) of this solution is measured for radioactivity by liquid scintillation counting. Next, 1/2000 cpm of labeled tet-1 is added. Then, 5 μl of cot-1 (10.0 μg / μl) and 0.2 μl of poly A (10.0 μg / μl) are added and incubated at 100 ° C. for 5 minutes.
[0200]
(Hybridization of ES cell mRNA probe with nucleic acid on microarray)
As described below, the probe prepared as described above is hybridized with the nucleic acid immobilized on the microarray prepared as described above.
[0201]
The membrane is subjected to prehybridization in 500 μl Perfect Hyb (Toyobo) at 68 ° C. for 30 minutes or more. Then incubate for 30 minutes at 68 ° C. in 250 μl fresh Perfect Hyb. Add the probe prepared above and incubate at 68 ° C overnight. It is then washed twice in 2 × SSC + 1% SDS for 15 minutes and twice in 0.1 × SSC + 1% SDS for 30 minutes. Dry the membrane and directly expose to high resolution IP.
[0202]
(Example 10; analysis using microarray)
Analysis on the microarray was performed as follows.
[0203]
1) Preparation of labeled cDNA
The animal under test is sacrificed and the tissue is frozen in liquid nitrogen. From the frozen tissue, total RNA is extracted using the following method.
[0204]
Extraction method of total RNA: Extraction of total RNA from frozen animal tissues according to guanidine (GTC) dissolution / cesium (CsCl) ultracentrifugation method (Chirgwin, JJ et al. 1979, Biochemistry 18, 5294). did.
[0205]
Using the prepared RNA and Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's manual, cDNA was synthesized as follows:

Was prepared, incubated at 65 ° C. for 5 minutes, and then left on ice for 2-3 minutes or more. next,

Was prepared, added to the above solution, and incubated at 42 ° C. for 1 hour. Next, the following was added.
[0206]
1.0 μl 10% SDS
1.0 μl 0.5 M EDTA
3.0 μl 3M NaOH
This was incubated at 68 ° C. for 30 minutes to degrade RNA. The tube was then returned to room temperature.
[0207]
Next, the following was added at room temperature to neutralize the reaction.
[0208]
10 μl 1 M Tris-HCl pH 7.5
3 μl 2N HCl.
[0209]
Free nucleotides (including radioactive ones) not incorporated into the cDNA were removed by a Sephadex® G50 (Amersham Biosciences Corp. Piscataway, NJ, USA) column.
[0210]
2) Hybridization on microarray
Hybridization on the microarray was performed as follows.
[0211]
Hybridization was performed using a hybridization oven (UVP HB-1000, UVP, Inc. Upland, CA, USA). For a 11.0 × 7.5 cm nylon membrane, 2.0 ml of hybridization buffer (750 mM NaCl; 50 mM NaH) ₂ PO ₄ 5 mM EDTA; 0.1% Ficoll; 0.1% PVP; 0.1% BSA; 120 μg / ml salmon sperm DNA; 10% dextran sulfate; 1.0% SDS) and a cDNA probe prepared by the above protocol. I went.
[0212]
Hybridization was performed at 68 ° C. for 24 hours.
[0213]
The following procedure was used for washing the nylon membrane (filter).
[0214]
(1) 2 × SSC, 55 ° C., 30 minutes
(2) 0.1 × SSC, 0.1% SDS, 65 ° C., 30 minutes.
[0215]
The detected hybridization signals were compared between the sources of labeled cDNA to identify those cDNAs that exhibited a specific expression pattern.
[0216]
(Analysis by computer)
Autoradiography was performed using BAS5000 (Fuji Film, Tokyo), and dot pattern analysis was performed using analysis software "ArrayGauge Ver. 1.2" (Fuji Film, Tokyo).
[0217]
(Example 11: Preparation of knockout animal)
In Example 10, spots that were expressed in the brain-derived cDNA screen and that did not show significant expression in the liver-derived cDNA screen were selected as showing a specific expression pattern. The schematic diagram is shown in FIG. ES cells corresponding to the spots identified to show a specific expression pattern were selected and microinjected into blastocysts of C57BL / 6 mice according to a conventional method. The blastocysts so treated were returned to the uterus of pseudopregnant mice. As a result, 6 mice (4 males and 2 females) with extremely high chimera ratio (having a very high ratio (90% or more) of cells derived from microinjected ES cell clones) were produced. Two males were used to breed with normal mice.
[0218]
F having a gene trap vector inserted into the chromosome ₁ Mice are selected, crossed, and homozygous for F with the gene trap vector. ₂ Prepare mice.
[0219]
(Example 12: Genetic analysis using knockout animal)
When two male mice with a chimera ratio of 90% or more were mated with normal C57 / BL6 female mice, the allele (allele) whose gene (GABA C receptor rho3 subunit gene) was trapped (disrupted) by the RET vector was changed to heterozygous. A number of mice were born. From the external observation, these heterozygous mice did not show any abnormal neurological findings. Thus, by breeding these hetero mice, a knockout animal having a homozygous allele in which the GABA C receptor rho subunit gene was disrupted by the RET vector was prepared.
[0220]
(Example 13: Production of knockout animal using known sequence)
Instead of synthesizing a DNA fragment by the 3′RACE method based on the sequence information determined in Example 8, a microarray was prepared using such sequence information. In such a case, a DNA fragment is synthesized based on the known sequence information. The synthesis uses an automated nucleic acid synthesizer. Alternatively, oligonucleotides can be synthesized directly on a microarray.
[0221]
2) Hybridization on microarray
Hybridization on the microarray was performed as follows.
[0222]
Hybridization was performed using a hybridization oven (UVP HB-1000, UVP, Inc. Upland, CA, USA). For a 11.0 × 7.5 cm nylon membrane, 2.0 ml of hybridization buffer (750 mM NaCl; 50 mM NaH) ₂ PO ₄ 5 mM EDTA; 0.1% Ficoll; 0.1% PVP; 0.1% BSA; 120 μg / ml salmon sperm DNA; 10% dextran sulfate; 1.0% SDS) and a cDNA probe prepared by the above protocol. I went.
[0223]
Hybridization was performed at 68 ° C. for 24 hours.
[0224]
The following procedure was used for washing the nylon membrane (filter).
[0225]
(1) 2 × SSC, 55 ° C., 30 minutes
(2) 0.1 × SSC, 0.1% SDS, 65 ° C., 30 minutes.
[0226]
The detected hybridization signals were compared between the sources of labeled cDNA to identify those cDNAs that exhibited a specific expression pattern.
[0227]
(Analysis by computer)
Autoradiography was performed using BAS5000 (Fuji Film, Tokyo), and dot pattern analysis was performed using analysis software "ArrayGauge Ver. 1.2" (Fuji Film, Tokyo).
[0228]
The detected hybridization signals were compared between the sources of labeled cDNA to identify those cDNAs that exhibited a specific expression pattern.
[0229]
As described above, the present invention has been exemplified by the preferred embodiments of the present invention. However, it is understood that the scope of the present invention should be construed only by the appended claims. Patents, patent applications, and literature cited herein are to be incorporated by reference in their entirety, as if the content itself were specifically described herein. Understood.
[0230]
【The invention's effect】
According to the present invention, a knockout mouse having desired properties can be produced much more easily than in the past. Further, it has become possible to identify a novel gene having desired properties, which cannot be identified by the conventional method.
[Brief description of the drawings]
FIG. 1 shows the structure of a retroviral vector (pRET gene trap vector) used in the present invention.
FIG. 1A shows the structure of the pRET vector in a packaging cell line. The 5 'LTR of the pRET vector has the wild-type sequence, while the 3' LTR does not have the transcription enhancer element of the U3 portion (this vector is a "self-inactivating" retroviral vector). Instead, the U3 portion of the 3 'LTR has a loxP signal.
FIG. 1B shows the structure of pRET in infected cells. The nucleotide sequence of the 3 'LTR is copied exactly into the 5' LTR during reverse transcription and insertion into the chromosome. Thus, the 5 'LTR in infected cells also lacks enhancers and has a loxP signal in the same orientation as the loxP signal in the 3' LTR. The pRET virus possesses three elements in the opposite direction of viral transcription: (i) a gene terminator cassette consisting of a strong splice acceptor (SA), multiple stop codons, an IRES sequence, GFP cDNA, and An efficient polyA signal; (ii) a complete and constitutive HSV tk cassette for virus titration and negative selection; and (iii) a splice donor (SD) and mRNA destabilization signal, Unique NEO cassette for enhanced poly A trap lacking poly A signal.
FIG. 1C shows the structure of the elements remaining on the chromosome after Cre-mediated excision. The hatched rectangle indicates the exon of the gene that was disrupted by the gene trap vector.
En (-): enhancer deletion; P1: MC1 promoter; P2: RNA polymerase II promoter (truncated form); X-GFP mRNA: fusion of 5 ′ half of gene X disrupted by gene trap vector with GFP cDNA Transcript.
FIG. 2 shows a schematic diagram of a DNA microarray chip used in the present invention.
FIG. 3 shows an example of analysis using the DNA microarray used in the present invention.
FIG. 4 is a diagram showing an example of a flow of a microarray analysis using cDNA obtained by the gene trap method of the present invention.
FIG. 5 shows an example of a method for producing a knockout animal used in the present invention.
FIG. 6 shows an example of a computer system used in the analysis of the present invention.

Claims (38)

A method for producing a knockout animal, comprising the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
3) obtaining genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe;
4) a step of selecting a gene having a desired property based on the genetic information; and 5) an embryo in which the gene having the desired property has been disrupted from the modified embryonic stem cells prepared in step 1) Selecting a sex stem cell, producing a knockout animal using the embryonic stem cells in which the gene has been disrupted,
A method comprising: A method for producing a knockout animal, comprising the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) a step of producing a knockout animal from the modified embryonic stem cells prepared in step 1);
3) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
4) obtaining the genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe; and 5) a gene having desired properties selected based on the genetic information; Correlating with the knockout animal,
A method comprising: 3. The method of claim 1 or 2, wherein the oligonucleotide or derivative thereof is identifiably labeled. The method according to claim 1, wherein the genetic information is analyzed using a microarray. 5. The method according to claim 4, wherein the microarray is a nylon membrane type. 3. The method according to claim 1 or 2, wherein the gene trap vector is in the form of a retrovirus vector or an improved poly A trap. The method according to claim 1 or 2, wherein the genetic information is an expression level of mRNA, a cell or tissue in which mRNA is expressed, an mRNA expression pattern, or regulation of mRNA expression. 3. The method of claim 1 or 2, wherein the desired property is to be responsive to antibiotic stimulation or to exhibit a significant expression pattern in cells or tissues. The desired properties are that the expression level is significantly increased or decreased with the canceration of the cell, the expression level is significantly increased with the induction of cell death, and specific in the hippocampus region that controls long-term memory in the brain. 3. The method of claim 1 or 2, wherein the method is selected from the group consisting of: The method according to claim 1, wherein the production of the knockout animal is performed by microinjection of embryonic stem cells into blastocysts. The method according to claim 1, wherein the oligonucleotide is a cDNA. A kit for producing a knockout animal,
1) a population of embryonic stem cells in which a gene has been gene-trapped; and 2) an oligonucleotide or a derivative thereof corresponding to the gene trapped in each embryonic stem cell in the population of embryonic stem cells,
A kit comprising: The kit according to claim 12, wherein the oligonucleotide or a derivative thereof is arranged on a microarray. 13. The kit according to claim 12, wherein the embryonic stem cells are provided in a frozen state. The kit according to claim 12, wherein the gene is derived from a rat, mouse or hamster. 13. The kit of claim 12, wherein said oligonucleotide or derivative thereof is about 150 to about 300 nucleotides in length. 14. The kit of claim 13, wherein the microarray comprises at least about 1000 oligonucleotides or derivatives thereof. 14. The kit of claim 13, wherein the microarray comprises about 1000-2000 oligonucleotides or derivatives thereof. The kit according to claim 13, wherein the oligonucleotide or a derivative thereof is a cDNA. A kit for producing a knockout animal,
1) Gene trap vector;
2) a population of embryonic stem cells; and 3) an oligonucleotide or a derivative thereof corresponding to a gene to be trapped in each embryonic stem cell in the population of embryonic stem cells;
A kit comprising: 21. The kit according to claim 20, wherein the oligonucleotide or derivative thereof is arranged on a microarray. A microarray on which an oligonucleotide or a derivative thereof is arranged, wherein the oligonucleotide or the derivative thereof is derived from a gene trapped in a gene-trapped embryonic stem cell used in a kit for producing a knockout animal. , Microarray. The gene-trapped embryonic stem cell, wherein the oligonucleotide or a derivative thereof corresponding to the gene trapped in the embryonic stem cell is arranged on a microarray used in a kit for producing a knockout animal. Sex stem cells. A system for producing a knockout animal, comprising:
1) Gene trap vector;
2) a population of embryonic stem cells;
3) an oligonucleotide or a derivative thereof corresponding to a gene to be trapped in each embryonic stem cell in the embryonic stem cell population; and 4) a means for analyzing the oligonucleotide or a derivative thereof;
A system comprising: 25. The system of claim 24, wherein the oligonucleotide or derivative thereof is disposed on a microarray, and wherein the means for analyzing is a means for analyzing a microarray. In addition, the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
3) obtaining genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe;
4) a step of selecting a gene having a desired property based on the genetic information; and 5) an embryo in which the gene having the desired property has been disrupted from the modified embryonic stem cells prepared in step 1) Selecting a sex stem cell, producing a knockout animal using the embryonic stem cells in which the gene has been disrupted,
25. The system of claim 24, comprising instructions indicating: In addition, the following steps:
1) Gene trapping using an embryonic stem cell and a gene trap vector to generate a population of modified embryonic stem cells;
2) a step of producing a knockout animal from the modified embryonic stem cells prepared in step 1);
3) preparing an oligonucleotide corresponding to the gene based on the sequence of the trapped gene in the modified embryonic stem cell;
4) obtaining the genetic information of the trapped gene using the prepared oligonucleotide or a derivative thereof as a probe; and 5) a gene having desired properties selected based on the genetic information; Correlating with the knockout animal,
25. The system of claim 24, comprising instructions indicating: A system for producing a knockout animal, comprising:
1) a population of gene-trapped embryonic stem cells;
2) an oligonucleotide or a derivative thereof corresponding to a gene trapped in each embryonic stem cell in the embryonic stem cell population; and 3) a means for analyzing the oligonucleotide or a derivative thereof;
A system comprising: 29. The system according to claim 28, wherein the oligonucleotide or derivative thereof is arranged on a microarray, and the means for analyzing is a means for analyzing a microarray. A knockout animal produced by the method according to claim 1. A nucleic acid molecule of a gene having desired properties, identified by the method according to any one of claims 1 to 11. A polypeptide of a gene having a desired property, identified by the method according to any one of claims 1 to 11. A recording medium for recording genetic information of a gene, obtained by the method according to any one of claims 1 to 11. The method according to claim 4, wherein the computer program causes a computer to execute a method for selecting embryonic stem cells for producing a knockout animal, the method comprising:
1) The address information of the oligonucleotides or derivatives thereof arranged in the microarray, and the genetics on the addresses in the microarray when an assay for providing the genetic information is performed using the microarray. Correlating with a signal indicating target information; and 2) correlating the address information with an ID of an embryonic stem cell from which the trapped gene is derived.
Wherein selection of genetic information indicative of a desired property indicates the ID of the corresponding embryonic stem cell,
Computer program. 5. The computer program for causing a computer to execute a method of determining which clone or gene in the population corresponds to a spot reflecting a desired property selected on the microarray, according to the method of claim 4. And the method comprises:
1) selecting a corresponding spot based on the input data of the desired property;
2) selecting a clone or gene correlated to the address of the selected spot;
3) displaying the selected clone or gene;
Including
Computer program. A computer-readable recording medium storing the computer program according to claim 34 or 35. 5. The method of claim 4, wherein the system performs a method of selecting embryonic stem cells for producing a knockout animal, wherein the system comprises:
A) a computer; and B) the computer program of claim 34.
Comprising,
system. The method according to claim 4, wherein a spot that reflects a desired property selected on the microarray corresponds to which clone or gene in the population,
A) a computer; and B) the computer program of claim 35.
Comprising,
system.

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